Targeting immunotherapy for amyloidosis

ABSTRACT

Disclosed are methods and compositions for targeting antibodies to amyloid deposits. For example, amyloid-reactive peptides that bind amyloid deposits are administered to a subject. Antibodies to the amyloid-reactive peptides are then administered to the subject. Upon administration of the antibodies, the amyloid-reactive peptides bind the antibodies and thus pre-target the antibodies to the amyloid deposits. In other examples, an amyloid-reactive fusion peptide contains an epitope of a known antibody. When the fusion peptide is administered to a subject, the fusion peptide binds amyloids in the subject. Administration to the subject of the known antibody that binds the epitope of the fusion peptide then targets the antibody to the amyloid deposit to which the fusion peptide is bound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit to U.S. Provisional PatentApplication No. 62/041,888, filed Aug. 26, 2014, which is titled“Pre-Targeting Immunotherapy for Amyloidosis.” The entire disclosure ofthe above-identified priority application is hereby fully incorporatedherein by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numberR01DK079984 awarded by National Institutes of Health. The government hascertain rights in the invention.

TECHNICAL FIELD

The present invention relates to antibodies that bind amyloid-reactivepeptides and amyloid-reactive fusion peptides, which in turn bindamyloid deposits. The combination of antibodies and amyloid-reactivepeptides may be used to treat multiple forms of amyloidosis by inducinga cellular response to clear amyloid deposits from the tissues ofaffected subjects.

BACKGROUND

Amyloidosis is a fatal protein-folding disorder characterized by theaggregation and deposition of proteinaceous fibrils and heparan sulfateproteoglycan in vital organs and tissues (Merlini, G. et al. (2003) N.Engl. J. Med. 349, 583-596; Merlini, G. et al. (2004) J. Intern. Med.255, 159-178; De Lorenzi, E. et al. (2004) Curr. Med. Chem. 11,1065-1084; Merlini, G. (2004) Neth. J. Med. 62, 104-105). Theunrelenting accumulation of amyloid invariably leads to organdysfunction and severe morbidity or death. The deposits can be cerebral,as in patients with Alzheimer's, Huntington's or prion diseases, orperipheral such as seen in patients with light chain (AL) amyloidosisand type 2 diabetes. Further sub-grouping into localized or systemicindicates whether the precursor protein is produced locally (at the siteof deposition) or circulates in the blood stream and deposits at distantanatomic sites, respectively (Westermark, P. et al. (2007) Amyloid. 14,179-183). Amyloid can affect any organ or tissue but the kidneys,pancreas, liver, spleen, nervous tissue and heart constitute the majorsites of deposition in patients with familial or sporadic forms ofperipheral amyloid disease. Alzheimer's disease currently affects morethan 4 million Americans and this figure is estimated to increase tomore than 16 million by the year 2050. It is by far the most common formof amyloidosis and poses the greatest socioeconomic impact. In contrast,the peripheral (or systemic) amyloidoses are orphan disorders butaccount for more than 5,000 new patients annually in the USA alone.

Of these, the major peripheral amyloidosis is light chain-associated(AL) amyloidosis, a sporadic monoclonal plasma cell dyscrasia resultingin the deposition of fibrils composed of immunoglobulin light chainproteins. AL accounts for approximately two thirds of all peripheralamyloid cases and has a calculated incidence of ˜1.4 per 100,000 personsper year in the USA, which is comparable to that of acute lymphocyticand chronic myeloid leukemia (Group, U. S. C. S. W. (2007) United StatesCancer Statistics: 1999-2003 Incidence and Mortality Web-Based Report,U.S. Department of Health and Human Services Centers for Disease Controland Prevention National Cancer Institute, Atlanta). Although AL is onefifth as common as the related plasma cell dyscrasia multiple myeloma itis arguably more devastating with a median survival of only 13.2 monthsdue partly to the rapidly progressive nature of the organ destruction,the lack of effective anti-amyloid therapeutics and the inability toeffectively diagnose the disease before organ failure occurs. Fewer than5% of all AL patients survive 10 years or more from the time ofdiagnosis (Comenzo, R. L. et al. (2002) Blood 99, 4276-4282). Moreover,in patients with cardiac AL amyloidosis the median survival is less than5 months.

Another prevalent form of peripheral amyloidosis in the U.S. isinflammation-associated (AA) amyloidosis, which is associated withchronic inflammatory disorders such as arthritis, tuberculosis andFamilial Mediterranean Fever. The incidence of AA is greatest in certainregions of Europe and the frequency varies among ethnic groups (Buck, F.S. et al. (1989) Mod. Pathol. 2, 372-377). In areas where FamilialMediterranean Fever is prevalent and goes untreated, the incidence of AAcan be 100%. In Europe the incidence, based on autopsy studies performedin the Denmark, is estimated to be 0.86% (Lofberg, H. et al. (1987) Actapathologica, microbiologica, et immunologica Scandinavica 95, 297-302);however, in patients with rheumatoid or psoriatic arthritis theoccurrence of AA can be as high as 26%. Such a high prevalence maywarrant a screening program to detect the disease earlier. Deposition ofamyloid is associated with a sustained increase in the plasmaconcentration of serum amyloid protein A (sAA), the precursor of theamyloid fibrils (Rocken, C. et al. (2002) Virchows Arch. 440, 111-122).AA differs from AL in the type of precursor protein that is depositedbut both share common mechanistic features associated with fibrilformation and deposition (Rocken, C. et al. (2006) J. Pathol. 210,478-487; Rocken, C. et al. (2001) Am. J. Pathol. 158, 1029-1038).

In addition to the disorders in which the etiopathology of amyloid iswell established, fibrillar deposits with the structural and tinctorialproperties of amyloid have been identified in other syndromes althoughtheir relevance to the disease state has yet to be established. In type2 diabetes for example, islet amyloid precursor protein (IAPP) depositsas amyloid in the Islets of Langerhans (Jaikaran, E. T. et al. (2001)Biochim. Biophys. Acta 1537, 179-203). The aggregation of IAPP resultsin oligomeric structures that are toxic to pancreatic cells (Lin, C. Y.et al. (2007) Diabetes 56, 1324-1332). Thus, it is suggested that theformation of IAPP amyloid in type 1 diabetic patients contributes to 13cell destruction and ushers in the transition to insulin dependence(Jaikaran, E. T. et al. (2001) Biochim. Biophys. Acta 1537, 179-203). Inanother example, plaques containing amyloid fibrils composed ofapolipoprotein A-I have been identified in over half of patients withatherosclerotic carotid arteries (Westermark, P. et al. (1995) Am. J.Pathol. 147, 1186-1192; Mucchiano, G. I. et al. (2001) J. Pathol. 193,270-275). The deposition of these fibrils was more common in olderpatients but apoA-I is undoubtedly present early in plaque development(Vollmer, E. et al. (1991) Virchows Arch. A. Pathol. Anat. Histopathol.419, 79-88). As a final example, Apo-A-I amyloid was also recentlyidentified in knee joint menisci obtained from patients having kneereplacement surgery and may contribute to the physical deterioration ofthe joint (Solomon, A. et al. (2006) Arthritis Rheum. 54, 3545-3550).

In total, more than 29 proteins have been chemically or serologicallyidentified as constituents of fibrils in amyloid deposits. It is thenature of these proteins that differentiate the diseases, determine thetreatment, and establish the prognosis. Although amyloid fibrils areassociated with a clinically heterogeneous group of diseases and canform from structurally distinct and functionally diverse precursorproteins, the deposits themselves share a number of remarkably similarcharacteristics including fibril structure, fibril epitopes and accrualof similar accessory molecules including heparan sulfate proteoglycans(HSPGs). Amyloid is a heterogeneous complex that includes, in additionto fibrils, glycosaminoglycans (GAGs) and in particular the perlecanHSPG (Ancsin, J. B. (2003) Amyloid 10, 67-79; Ailles, L. et al. (1993)Lab. Invest. 69, 443-448; Kisilevsky, R. (1994) Mol. Neurobiol. 9,23-24; Kisilevsky, R. (1990) Lab. Invest. 63, 589-591; Snow, A. D. etal. (1987) Lab. Invest. 56, 120-123; Li, J. P. et al. (2005) Proc. Natl.Acad. Sci. USA 102, 6473-6477). A partial list of amyloid and amyloidrelated disorders is provided in Table 1 (below).

TABLE 1 Partial List of Amyloid and Amyloid-Related Disorders Aquired(A)/ Systemic (S) or Herditary (H)/ Amyloid type Precursor Localized (L)Organs Syndrome AL Immunoglobulin light chain S, L A/All but *CNSPrimary, Myleoma AH Immunoglobulin heavy chain S, L A/All but CNS Aβ₂Mβ₂-microglobulin S, A/Musculoskeletal Hemodialysis ATTR Transthyretinvariants S, L A/Heart, Familial, Senile tenosynovium systemic Wild typeTransthyretin S H/heart, eye, Aging TTR leptomen AA Serum amyloidprotein A S A/All but CNS Reactive, chronic inflammation AApoAIApolipoprotein AI S H/heart, liver etc Familial AApoAII ApolipoproteinAII S H/Kidney AGel Gelsolin S H/PNS, cornea Familial ALys Lysozyme SH/Kidney Familial ALect2 leukocyte chemotactic factor S A/Kidney Renalamyloid 2 AFib Fibrinogen α variants S H/Kidney Familial ACys Cystatinvariants S H/*PNS, skin ACal (Pro)calcitonin L A/Thyroid Thyroid tumorsAMed Lactadherin L A/Senile aortic Aging media AIAPP Islet amyloidpolypeptide L A/Islets of Type 2 diabetes Langerhans APro Prolactin LA/Pituitary Aging pituitary AIns Insulin L A/Injection site IatrogenicAPrP Prion protein L A/H/, brain Spongiform encephalopathies Aβ Aβprecursor protein L A/H/brain Alzheimer's disease and aging *PNS =peripheral nervous system; CNS = central nervous system

To date, the most effective therapeutic intervention for removingamyloid deposits, which may promote recovery of organ function and leadto an improved prognosis, involves the use of amyloid-reactiveantibodies as a means of immunotherapy. Several immunotherapies(antibodies) have been developed for amyloid-related diseases, includingmonoclonal antibody 11-1F4 for the treatment of AL amyloidosis, NEOD001for patients with AL amyloidosis, GSK2398852 (anti-SAP monoclonalantibody) for amyloidosis, Solanezumab for Alzheimer's disease,intravenous IgG (WIG) for Alzheimer's disease, and Bapineuzumab forAlzheimer's disease. Each of these approaches has limitations or has notbeen validated in extensive clinical trials (Phase 2/3).

SUMMARY

In certain example aspects, provided are amyloid-reactive peptides thatbind amyloid deposits. For example, the amyloid-reactive peptides bindone or more of the amyloids identified in Table 1. In certain exampleembodiments, provided are amyloid-reactive fusion peptides that bindamyloid deposits. The amyloid-reactive fusion peptides are fused, forexample, to an epitope of a known antibody. In certain example aspects,provided are antibodies that bind the amyloid-reactive peptides. Alsoprovided are the antibodies, including the amyloid-reactive antibodies,that bind the epitope of amyloid-reactive fusion peptides.

In certain example aspects, provided is a method of targeting an amyloiddeposit for clearance. The method includes, for example, contacting anamyloid deposit with an amyloid-reactive peptide that binds amyloiddeposits. The method also includes contacting the amyloid-reactivepeptide with an antibody that binds the amyloid-reactive peptide.Contacting the amyloid-reactive peptide with the antibody that binds theamyloid-reactive peptide pre-targets the amyloid deposit for clearance.Thereafter, pre-targeting of the amyloid deposit results in clearance ofthe deposit.

In certain example aspects, provided is a method for clearing amyloiddeposits in a subject. The method includes, for example, selecting asubject having amyloidosis and administering to the subject anamyloid-reactive peptide that binds to the amyloid deposits. In additionto administering amyloid-reactive peptide, the method includesadministering to the subject an antibody, or a functional group orfragment thereof, which binds to the amyloid-reactive peptide.Administering the antibody or functional fragment thereof to the subjectresults in clearance of the amyloid deposit in the subject, therebytreating the subject.

In certain example aspects, the amyloid-reactive peptide includes anepitope bound to the amyloid-reactive peptide. The epitope, for example,is an epitope of a known antibody. When administered to a subject, forexample, binding of the antibody or functional fragment thereof to theepitope results in increased clearance of the amyloid deposit. Incertain example aspects, the epitope includes a motif for binding anamyloid-reactive antibody. For example, the antibody is anamyloid-reactive antibody and thus can bind the epitope of the amyloiddeposit directly.

In certain example aspects, provided is a method for clearing amyloiddeposits in a subject. The method includes, for example, selecting asubject with amyloidosis and administering to the subject an effectiveamount of an amyloid-reactive fusion peptide. The amyloid-reactivefusion peptide comprises an amyloid-reactive peptide that binds toamyloid deposits and an epitope fused to the amyloid-reactive peptidethat binds an antibody. The method also includes administering to thesubject an effective amount of the antibody or fragment thereof Bindingof the antibody or fragment thereof to the amyloid-reactive fusionpeptide results in clearance of the amyloid deposit.

In certain example aspects, provided is a kit. The kit includes, forexample, an effective amount of the amyloid-reactive peptides or fusionpeptides and an effective amount of antibodies that bind theamyloid-reactive peptides or fusion peptides. The kit also optionallyincludes instructions for using the kit, such as for administering theamyloid-reactive peptides, fusion peptides, and antibodies as describedherein.

In certain example aspects, provided is a substantially pure antibodyhaving binding affinity for an amyloid-reactive peptide, such as theamyloid-reactive peptides identified in Table 2.

These and other aspects, objects, features and advantages of the exampleembodiments will become apparent to those having ordinary skill in theart upon consideration of the following detailed description ofillustrated example embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1B are schematic drawings showing targeting of antibodies toamyloids via amyloid-reactive peptides (e.g., p5 or p5+14) (FIG. 1A) andvia an antibody epitope (e.g., peptide A12) bound to theamyloid-reactive peptide (e.g., p5 or 05+14) (FIG. 1B), in accordancewith certain example embodiments.

FIG. 2 is a graph demonstrating targeting of monoclonal antibody clones4, 5, 12, and 13 to synthetic light chain-associated (AL) fibrilscomposed of the 2,6 variable domain (rVλ6Wil, aka WIL), coated withpeptide p43 or p5+14, in accordance with certain example embodiments.

FIG. 3 is a graph demonstrating targeting of antibody clones 4, 5, 12,and 13 to murine AA amyloid-associated amyloid extract (AEF), coatedwith peptide p43 or p5+14, in accordance with certain exampleembodiments.

FIG. 4 is a graph demonstrating that monoclonal antibody clones 4, 5,12, and 13 are capable of capturing biotinylated peptide p5+14 fromsolution, in accordance with certain example embodiments.

FIG. 5 is a graph showing the comparison of pre-targeting on AA AEF vs.standard one-step binding of pre-incubated complex, in accordance withcertain example embodiments.

FIG. 6 is a series of micrographs demonstrating that peptide p5+14co-localizes with amyloid deposits (left column), which are alsoobserved in the Congo red-stained tissue section (right column), inaccordance with certain example embodiments.

FIG. 7 is a series of micrographs comparing the binding of monoclonalantibody clones 4, 5, 12, and 13 to amyloid deposits in the presence andabsence of peptide, in accordance with certain example embodiments.

FIG. 8 is a schematic drawing showing two predicted structures of p66based on the amino acid sequence of p66, in accordance with certainexample embodiments.

FIG. 9 is a graph showing 11-1F4 binding to p66 (11-1F4 peptope) ascompared to known 11-1F4 epitope peptides [Len(1-22)], therebydemonstrating that the epitope portion of p66 is not compromised by thepresence of the p5+14 sequence, in accordance with certain exampleembodiments.

FIG. 10 is a graph demonstrating that 11-1F4 binds both Wil and Aβ(1-40)amyloid fibrils with low affinity, in accordance with certain exampleembodiments.

FIG. 11 is a graph demonstrating that the reactivity of the 11-1F4monoclonal antibody is enhanced, particularly to Wil fibrils but also tothe Aβ(1-40) fibrils, in the presence of p66, in accordance with certainexample embodiments.

FIG. 12 is a graph demonstrating that fibril material is required forbinding of p66 and hence for p66-mediated targeting of antibodies, inaccordance with certain example embodiments.

FIG. 13 is a pair of graphs showing that peptide p66 and p5+14 bindequally well to synthetic and naturally-occurring amyloid samples in0.15 M NaCl (FIG. 13A) and in 1.0 M NaCl (FIG. 13B), in accordance withcertain example embodiments.

FIG. 14 is a series of microautoradiographs and Congo-red-stainedmicrographs demonstrating that p66 injected into mice selectively bindsamyloid deposits in a variety of tissues in vivo, in accordance withcertain example embodiments.

FIG. 15 is a series of microautoradiographs demonstrating that p66injected into healthy, wild-type mice does not bind to any of thetissues examined in vivo, in accordance with certain exampleembodiments.

FIG. 16 is an image (FIG. 16A) and a graph (FIG. 16B) showing SPECT/CTimaging of ¹²⁵I-p66 in AA mice at 4 and 72 h post injection (pi) (FIG.16A) and tissue biodistribution of ¹²⁵I-p66 in AA and WT (healthy,amyloid-free) mice at 2 h post injection (pi) (FIG. 16B) in vivo, inaccordance with certain example embodiments.

FIG. 17 is a series of micrographs and microautoradiographs from varioustissue types showing evaluation of mice at 24 h post injection of 11-1F4monoclonal antibody into AA mice pre-targeted with peptope p66 in vivo,in accordance with certain example embodiments. Peptide p66 is shownco-localizing with ¹²⁵I-11-1F4 monoclonal antibody and AA amyloid.

FIG. 18 is a series of microautoradiographs from various tissue typesshowing evaluation of mice at 24 h post injection of ¹²⁵1-11-1F4monoclonal antibody into AA mice pre-targeted with p5+14 controlpeptide, in accordance with certain example embodiments. The 11-1F4monoclonal antibody does not localize to amyloids when the mice arepre-treated with p5+14 control peptide alone.

FIG. 19 is a series of micrographs showing evaluation of livermacrophages in AA mice at 72 h post injection of 11-1F4 monoclonalantibody pre-injected with p66 or p5+14, in accordance with certainexample embodiments. The combination of p66 with 11-1F4 monoclonalantibody results in increased macrophage accumulation in the liveraround amyloid deposits.

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

Overview

Described herein are compositions that include amyloid-reactivepeptides, amyloid-reactive fusion peptides, and antibodies toamyloid-reactive peptides and fusion peptides. Also described herein aremethods of using the same for the treatment of amyloidosis. For example,the amyloid-reactive peptides, amyloid-reactive fusion peptides, andantibodies may be used to target amyloid deposits in subjects withamyloidosis. By targeting amyloid deposits in the subject, theamyloid-reactive peptides, amyloid-reactive fusion peptides, antibodies,and methods described herein initiate clearance of the amyloid depositsby the subject's own immune system. That is, the amyloid-reactivepeptides, amyloid-reactive fusion peptides, antibodies, and methodsdescribed thus treat the subject having amyloidosis.

More particularly, amyloid-reactive peptides and fusion peptides areprovided that bind to one or more components of the amyloid (e.g.,protein fibrils or glycosaminoglycans) that make up an amyloid deposit.For example, the amyloid-reactive peptides and fusion peptides may bepan amyloid-reactive peptides and fusion peptides that bind to multipleamyloid deposit types.

Also provided are antibodies that bind amyloid-reactive peptides andfusion peptides. For example, the antibodies are raised against one ormore of the amyloid-reactive peptides such that the antibodies bind tothe one or more amyloid-reactive peptides. When one or more of theamyloid-reactive peptides are administered to a subject, for example,the amyloid-reactive peptides localize to—and bind to—amyloid depositswithin the subject. Thereafter, when the antibodies are administered tothe subject, the antibodies bind to the amyloid-reactive peptides. Assuch, the antibodies bind to the amyloid deposit indirectly via theamyloid-reactive peptides.

Additionally or alternatively, in certain examples the amyloid-reactivepeptide is fused to an “epitope” peptide of a known (corresponding)antibody to form an amyloid-reactive fusion peptide. For example, theepitope may be fused to the C-terminal end of the amyloid-reactivepeptide. With the fused epitope, the antibody recognizes and binds tothe peptide epitope (the “peptope”) of the amyloid-reactive peptide.When such peptope-containing amyloid-reactive fusion peptides areadministered to a subject, for example, the peptides localize and bindto amyloid deposits within the subject. Administration of the antibodiesto the subject then results in binding of the antibodies to theamyloid-reactive fusion peptides via the peptope.

Additionally or alternatively, in certain examples the epitope potion ofthe amyloid-reactive peptide is a known epitope of an amyloid-reactiveantibody. That is, the antibody is known to bind one or more amyloidproteins. Hence, fusion of the amyloid-reactive antibody epitope to theamyloid-reactive peptide allows binding of amyloid-reactive antibody tothe amyloid-reactive fusion peptide via the amyloid-reactive antibodyepitope. When such amyloid-reactive fusion peptides containingamyloid-reactive antibody epitopes are administered to a subject, thepeptides localize and bind to amyloid deposits within the subject.Administration of the amyloid-reactive antibodies to the subject thenresults in binding of the amyloid-reactive antibodies to theamyloid-reactive fusion peptides via the peptope. Further, because theantibody is an amyloid-reactive antibody, the antibody also binds toamyloid deposits directly.

In such fusion peptide examples, the ability of the pan amyloid-reactivefusion peptides to bind to all or a subset of amyloid deposit typesallows administration of a single antibody to be effective againstmultiple amyloid deposit types. In other words, while theamyloid-reactive antibody may only bind one or a few amyloid types, useof the amyloid-reactive fusion peptides (that include theamyloid-reactive antibody epitope) can pre-target the amyloid-reactiveantibody to multiple amyloid types. Use of the amyloid-reactiveantibodies has the additional advantage of directly targeting theamyloid deposits to which the amyloid-reactive antibodies are reactive.

Without wishing to be bound by any particular theory, it is believedthat binding of the amyloid-reactive antibodies to the amyloiddeposit—via binding to the amyloid-reactive peptides or fusion peptidesas described herein—results in clearance of the amyloid deposits, forexample, through processes such as opsonization and phagocytosis. Inother words, it is believed that localization of antibodies to amyloiddeposits triggers an immune response in which opsonization and/orphagocytosis remove all or part of the targeted amyloid deposits. Byinitiating and facilitating clearance of amyloid deposits in a subjectsuffering from amyloidosis, the amyloid-reactive peptides,amyloid-reactive fusion peptides, antibodies, and methods describedherein may be used to treat the subject. Further, by using knownamyloid-reactive antibodies to bind to the amyloid-reactive fusionpeptides, the methods and compositions described herein allow existingantibodies, including non-amyloid-specific antibodies, to be adapted foruse in the treatment of amyloidosis.

Summary of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes IX, published by Jones and Bartlet,2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0632021829); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 9780471185710) and other similarreferences. As used herein, the singular forms “a,” “an,” and “the,”refer to both the singular as well as plural, unless the context clearlyindicates otherwise. The abbreviation, “e.g.” is derived from the Latinexempli gratia, and is used herein to indicate a non-limiting example.Thus, the abbreviation “e.g.” is synonymous with the term “for example.”As used herein, the term “comprises” means “includes.” All publications,patent applications, patents, and other references mentioned herein areexpressly incorporated herein by reference in their entirety.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. In case of conflict,the present specification, including explanations of terms, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting. To facilitate reviewof the various example embodiments of this disclosure, the explanationsof specific terms are provided below.

An “amyloid-reactive peptide” is a peptide that binds to amyloiddeposits, such as any of the amyloids identified in Table 1. Theamyloid-reactive peptide may also be a “pan” amyloid binding peptide,meaning that the amyloid-reactive peptide binds to multiple amyloidtypes. An “amyloid-reactive fusion peptide,” for example, isamyloid-reactive peptide that is fused to another peptide, such as anepitope, resulting in a fusion peptide.

“Administration” or “administering” refers to the introduction of acomposition into a subject by a chosen route. For example, if the chosenroute is intravenous, the composition is administered by introducing thecomposition into a vein of the subject. In some examples, the peptidesand antibodies disclosed herein are administered to a subject.

“Animal” refers to living multi-cellular vertebrate organisms, acategory that includes, for example, mammals and birds.

“Antibody” refers to single chain, two-chain, and multi-chain proteinsand glycoproteins belonging to the classes of polyclonal, monoclonal,chimeric and hetero immunoglobulins (monoclonal antibodies beingpreferred); it also includes synthetic and genetically engineeredvariants of these immunoglobulins. An “antibody fragment” includes Fab,Fab′, F(ab′)2, and Fv fragments, as well as any portion of an antibodyhaving specificity toward a desired target epitope or epitopes. A“monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes. Monoclonal antibodies are produced by methods known tothose of skill in the art, for instance by making hybridantibody-forming cells from a fusion of myeloma cells with immune spleencells.

“Epitope” refers to a site on an antigen recognized by an antibody, asdetermined by the specificity of the antibody amino acid sequence.Epitopes are also called antigenic determinants. For example, theepitope may be portion of a recombinant protein that is recognized bythe particular antibody. Further, the epitope may be a conformationalepitope and linear epitope.

“Chimeric antibody” refers to an antibody that includes sequencesderived from two different antibodies, which typically are of differentspecies. Most typically, chimeric antibodies include human and murineantibody fragments, generally human constant and murine variableregions.

“Humanized antibody” refers to an antibody derived from a non-humanantibody, typically murine, and a human antibody which retains orsubstantially retains the antigen-binding properties of the parentantibody but which is less immunogenic in humans.

“Complementarity Determining Region,” or CDR refers to amino acidsequences that together define the binding affinity and specificity ofthe natural Fv region of a native immunoglobulin binding site. The lightand heavy chains of an immunoglobulin each have three CDRs, designatedL-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Bydefinition, the CDRs of the light chain are bounded by the residues atpositions 24 and 34 (L-CDR1), 50 and 56 (L-CDR2), 89 and 97 (L-CDR3);the CDRs of the heavy chain are bounded by the residues at positions 31and 35b (H-CDR1), 50 and 65 (H-CDR2), 95 and 102 (H-CDR3), using thenumbering convention delineated by Kabat et al., (1991) Sequences ofProteins of Immunological Interest, 5th Edition, Department of Healthand Human Services, Public Health Service, National Institutes ofHealth, Bethesda (NIH Publication No. 91-3242).

“Framework region” refers to amino acid sequences interposed betweenCDRs. These portions of the antibody serve to hold the CDRs in anappropriate orientation for antigen binding.

“Specificity Determining Residue,” or SDR refers to amino acid residuesof an immunoglobulin that are directly involved in antigen contact.

“Constant region” refers to the portion of the antibody molecule thatconfers effector functions. In the present invention, the variantantibodies include constant regions derived from human immunoglobulins.The heavy chain constant region can be selected from any of fiveisotypes: alpha, delta, epsilon, gamma or mu. Heavy chains of varioussubclasses (such as the IgG subclass of heavy chains) are responsiblefor different effector functions. Thus, by choosing the desired heavychain constant region, humanized antibodies with the desired effectorfunction can be produced. The light chain constant region can be of thekappa or lambda type, preferably the kappa type.

“Amino acid” or “amino acid residue” refers to any naturally occurringamino acid, any non-naturally occurring amino acid, any modifiedincluding derivatized amino acid, or any amino acid mimetic known in theart. The amino acid may be referred by both their common three-letterabbreviation and single letter abbreviation.

The terms “amyloids,” “amyloid deposits,” or “amyloid fibrils” refer toinsoluble fibrous protein aggregates sharing specific structural traits.Abnormal accumulation of amyloids in organs may lead to amyloidosis.Although they are diverse in their occurrence, all amyloids have commonmorphologic properties such as stain with specific dyes such as Congored, and have a characteristic red-green birefringent appearance inpolarized light after staining Amyloids also share commonultrastructural features and common x-ray diffraction and infraredspectra.

“Amyloidosis” refers to a pathological condition or diseasecharacterized by the presence of amyloids, such as the presence ofamyloid deposits.

The terms “clear” or “clearance” refer to reducing or removing by ameasurable degree. For example, the clearance of an amyloid deposit asdescribed herein relates to reducing or removing the deposit to ameasurable or discernable degree.

“Carrier” refers to conventional pharmaceutically acceptable carriers.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 19^(th) Edition (1995), for example, describescompositions and formulations suitable for pharmaceutical delivery ofthe peptides disclosed herein. In general, the nature of the carrierwill depend on the particular mode of administration being employed. Forinstance, parenteral formulations usually comprise injectable fluidsthat include pharmaceutically and physiologically acceptable fluids suchas water, physiological saline, balanced salt solutions, aqueousdextrose, glycerol or the like as a vehicle. For solid compositions(e.g., powder, pill, tablet, or capsule forms), conventional non-toxicsolid carriers can include, for example, pharmaceutical grades ofmannitol, lactose, starch, or magnesium stearate. In addition tobiologically neutral carriers, pharmaceutical compositions to beadministered can contain minor amounts of non-toxic auxiliarysubstances, such as wetting or emulsifying agents, preservatives, and pHbuffering agents and the like, for example sodium acetate or sorbitanmonolaurate.

Example carriers include excipients or stabilizers that are nontoxic tothe cell, tissue, mammal, or subject being exposed thereto at thedosages and concentrations employed. Often the pharmaceuticallyacceptable carrier is an aqueous pH buffered solution. Examples ofpharmaceutically acceptable carriers also include, without limitation,buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween®, polyethylene glycol (PEG), and Pluronics®. As usedherein, a chimeric antibody refers to an antibody which includessequences derived from two different antibodies, which typically are ofdifferent species. Most typically, chimeric antibodies include human andmurine antibody fragments, generally human constant and murine variableregions.

“DNA” (deoxyribonucleic acid) refers to a long chain polymer whichconstitutes the genetic material of most living organisms (some viruseshave genes composed of ribonucleic acid (RNA)). The repeating units inDNA polymers are four different nucleotides, each of which contains oneof the four bases, adenine, guanine, cytosine and thymine bound to adeoxyribose sugar to which a phosphate group is attached. Triplets ofnucleotides (referred to as codons) code for each amino acid in apolypeptide. The term codon is also used for the corresponding (andcomplementary) sequence of three nucleotides in the mRNA that istranscribed from the DNA.

“Effective amount” or “suitable amount” or “therapeutically effectiveamount” refers to an amount of a substance sufficient to effect thebeneficial or desired clinical or biochemical results. An effectiveamount can be administered one or more times. For example, an effectiveamount of a peptide or fusion peptide as described herein is an amountthat is sufficient to bind to and allow detection of the amyloids. Apeptide or fusion peptide as described herein may be effective, forexample, when parenterally administered in amounts above about 1 μg perkg of body weight to about 30 mg/kg. A theirapeutically effective amountof an antibody described herein is the amount that is sufficient to bindthe peptide or fusion peptide as described herein.

“Immune cell” refers to any cell involved in a host defense mechanism.These can include, for example, T cells, B cells, natural killer cells,neutrophils, mast cells, macrophages, antigen-presenting cells,basophils, eosinophils, and neutrophils. An “immune response” is aresponse of a cell of the immune system, such as a macrophage,neutrophil, a B cell, or a T cell, to a stimulus.

“Label” refers to a detectable compound or composition that isconjugated directly or indirectly to another molecule to facilitatedetection of that molecule. Specific, non-limiting examples of labelsinclude fluorescent tags, chemiluminescent tags, haptens, enzymaticlinkages, and radioactive isotopes.

A “mammal” refers to any animal classified as a mammal, includinghumans, domestic and farm animals, and zoo, sports, or pet animals, suchas dogs, cats, cattle, horses, sheep, pigs, and so on. The mammal may bea human.

“Operably linked” refers to a first nucleic acid sequence that isconnected to a second nucleic acid sequence when the first nucleic acidsequence is placed in a functional relationship with the second nucleicacid sequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

“Opsonize” or “opsonization,” as used herein, refer toimmunoglobulin-based recognition of a target as “foreign” by the host'scellular immune system. For example, the binding of an immunoglobulin,such as the antibodies described herein, to an amyloid-deposit via anamyloid-reactive peptide or fusion peptide enhances the phagocytizationof amyloid fibrils.

“Peptide” refers to any peptide or peptidomimetic structure comprisingor consisting of two or more amino acids, including chemicalmodifications and derivatives of amino acids. For example, the peptidemay be modified to include epitope capable of binding an antibody. Incertain example embodiments, a peptide may be an amyloid-reactivepeptide, meaning that the peptide reacts with an amyloid by binding tothe amyloid.

“Polypeptide” refers to a polymer in which the monomers are amino acidresidues that are joined together through amide bonds. When the aminoacids are alpha-amino acids, either the L-optical isomer or theD-optical isomer can be used, the L-isomers being preferred. The terms“polypeptide” or “protein” as used herein is intended to encompass anyamino acid sequence and include modified sequences such asglycoproteins. The term “polypeptide” is specifically intended to covernaturally occurring proteins, as well as those that are recombinantly orsynthetically produced. In some examples, a polypeptide is one or moreof the peptides disclosed herein. As used herein, the terms “fusionprotein” or “fusion polypeptide” or “fusion peptide” refer to anon-naturally occurring protein having the portion of the peptide andanother portion that has been added to the protein. For example, anantibody epitope may be covalently bound to the protein to form a fusionprotein.

“Protein” refers to a biological molecule encoded by a gene andcomprised of amino acids.

“Pharmaceutical agent” refers to a chemical compound or compositioncapable of inducing a desired therapeutic or prophylactic effect whenproperly administered to a subject or a cell. For example, apharmaceutical agent may include a peptide as described herein and anantibody described herein, the administration of which result inclearance of an amyloid deposit.

“Purified” or “isolated” molecule refers to biological or syntheticmolecules that are removed from their natural environment and areisolated or separated and are free from other components with which theyare naturally associated. The term “purified” does not require absolutepurity; rather, it is intended as a relative term. Thus, for example, apurified or “substantially pure” protein preparation is one in which theprotein referred to is more pure than the protein in its naturalenvironment within a cell or within a production reaction chamber (asappropriate).

“Recombinant” nucleic acid is one that has a sequence that is notnaturally occurring or has a sequence that is made by an artificialcombination of two otherwise separated segments of sequence. Thisartificial combination is often accomplished by chemical synthesis or,more commonly, by the artificial manipulation of isolated segments ofnucleic acids, e.g., by genetic engineering techniques.

The term “specifically binds” refers to a non-random binding reactionbetween two molecules, for example between a peptide of the presentinvention and an amyloid. The term “specifically binds” may be usedinterchangeably with “selectively targets” or “selectively associates.”

The term “selectively targets” or “selectively associates” withreference to amyloids, refers to, for example, the selectivelocalization or binding to the amyloid. For example, an amyloid-reactivepeptide or fusion peptide as described herein pre-targets an amyloiddeposit by binding to the deposit. An antibody binding the peptide orfusion peptide then targets the amyloid, such as for opsinization, asdescribed herein.

“Sequence identity” refers to the similarity between two nucleic acidsequences, or two amino acid sequences, and is expressed in terms of thesimilarity between the sequences, otherwise referred to as sequenceidentity. Sequence identity is frequently measured in terms ofpercentage identity (or similarity or homology); the higher thepercentage, the more similar the two sequences are.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman Adv. Appl. Math. 2: 482, 1981; Needleman & Wunsch J. Mol. Biol.48: 443, 1970; Pearson & Lipman Proc. Natl. Acad. Sci. USA 85: 2444,1988; Higgins & Sharp Gene 73: 237-244, 1988; Higgins & Sharp CABIOS 5:151-153, 1989; Corpet et al. Nuc. Acids Res. 16, 10881-90, 1988; Huanget al. Computer Appls. In the Biosciences 8, 155-65, 1992; and Pearsonet al. Meth. Mol. Bio. 24, 307-31, 1994. Altschul et al. (J. Mol. Biol.215:403-410, 1990), presents a detailed consideration of sequencealignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J.Mol. Biol. 215:403-410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.

A “subject” refers to a vertebrate. The vertebrate may be a mammal, forexample, a human. The subject may be a human patient. A subject may be apatient suffering from or suspected of suffering from a disease orcondition and may be in need of treatment or diagnosis or may be in needof monitoring for the progression of the disease or condition. Thepatient may also be in on a treatment therapy that needs to be monitoredfor efficacy. In some example embodiments, a subject includes a subjectsuffering from amyloidosis, such as Alzheimer's, Huntington's or priondiseases, or peripheral amyloidosis such as seen in patients with lightchain (AL) amyloidosis and type 2 diabetes.

The terms “treating” or “treatment” refer to a therapeutic interventionthat ameliorates a sign or symptom of a disease or pathologicalcondition after it has begun to develop. The term “ameliorating,” withreference to a disease or pathological condition, refers to anyobservable beneficial effect of the treatment. The beneficial effect canbe evidenced, for example, by a delayed onset of clinical symptoms ofthe disease in a susceptible subject, a reduction in severity of some orall clinical symptoms of the disease, a slower progression of thedisease, an improvement in the overall health or well-being of thesubject, or by other parameters well known in the art that are specificto the particular disease. A “prophylactic” treatment is a treatmentadministered to a subject who does not exhibit signs of a disease orexhibits only early signs for the purpose of decreasing the risk ofdeveloping pathology.

A “vector” refers to a nucleic acid molecule as introduced into a hostcell, thereby producing a transformed host cell. Recombinant DNA vectorsare vectors having recombinant DNA. A vector can include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector can also include one or more selectable markergenes and other genetic elements known in the art. Viral vectors arerecombinant DNA vectors having at least some nucleic acid sequencesderived from one or more viruses. The term vector includes plasmids,linear nucleic acid molecules, and as described throughout adenovirusvectors and adenoviruses.

Amyloid-Reactive Peptides

In certain example embodiments, provided are amyloid-reactive peptidesand amyloid-reactive fusion peptides that specifically bind amyloids andthus are useful in the various methods and pharmaceutical compositionsdescribed herein. As “amyloid-reactive” peptides, the peptides bind toand interact with amyloids and/or components of amyloid deposits. Forexample, the amyloid-reactive peptides and fusion peptides bind one ormore components of the fibrils that make up an amyloid deposit. Theamyloid type can be any amyloid.

Additionally or alternatively, the amyloid-reactive peptides and fusionpeptides may bind one or more other amyloid deposit components, such asheparan sulfate proteoglycans and glycosaminoglycans (GAGs). In certainexample embodiments, the amyloid-reactive peptides and fusion peptidesare synthetic pan amyloid-reactive peptides that bind to multipleamyloid deposit types. For example, the amyloid-reactive peptides andfusion peptides may bind any one of AA, AL, AH, ATTR, Aβ2M, ALect2, Wildtype TTR, AApoAI, AApoAII, AGel, ALys, ALect2, Afib, ACys, ACal, AMedin,AIAPP, APro, AIns, APrP, AP, or combinations thereof or other amyloids.In certain example embodiments, the amyloid-reactive peptide is apeptide disclosed in U.S. Pat. No. 8,808,666, which is expresslyincorporated herein by reference in its entirety.

Additionally or alternatively, the amyloid-reactive peptides and fusionpeptides may include a functional fragment that binds one or moreamyloid types. Such fragments, for example, maintain the amyloid bindingcharacteristics of the parent amyloid-reactive peptide. In certainexample embodiments, one or more of the amyloid-reactive peptides andfusion peptides described herein bind to multiple amyloid deposit types.For example, the fragment of the amyloid-reactive peptide may be a panamyloid-reactive peptide fragment that binds to multiple amyloid types.

The amyloid-reactive peptides include, for example, from about 3 toabout 55 amino acids. For example, the peptides may include about 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino acids.In certain example embodiments, the peptides may have a molecular weightof between about 200 Da to about 6 kDa. The molecular weight of thepeptides may be about 300 Da, 400 Da, 500 Da, 1 Kda, 2 kDa, 3 kDa, 4kDa, or 5 kDa, for example.

In certain example embodiments, the amino acids forming all or a part ofthe amyloid-reactive peptides and fusion peptides described herein maybe stereoisomers. Additionally or alternatively, the amino acids formingall or a part of the peptides described herein may be modifications ofnaturally occurring amino acids, non-naturally occurring amino acids,post-translationally modified amino acids, enzymatically synthesizedamino acids, derivatized amino acids, constructs or structures designedto mimic amino acids, and the like. The amino acids forming the peptidesof the present invention may be one or more of the 20 common amino acidsfound in naturally occurring proteins, or one or more of the modifiedand unusual amino acids. In certain example embodiments, the amino acidsmay be D- or L-amino acids.

In certain example embodiments, the peptides may also include one ormore modified amino acids. The modified amino acid may be a derivatizedamino acid or a modified and unusual amino acid. Examples of modifiedand unusual amino acids include but are not limited to, 2-Aminoadipicacid (Aad), 3-Aminoadipic acid (Baad), β-Amino-propionic acid (Bala,β-alanine), 2-Aminobutyric acid (Abu, piperidinic acid), 4-Aminobutyricacid (4Abu), 6-Aminocaproic acid (Acp), 2-Aminoheptanoic acid (Ahe),2-Aminoisobutyric acid (Aib), 3-Aminoisobutyric acid (Baib),2-Aminopimelic acid (Apm), 2,4-Diaminobutyric acid (Dbu), Desmosine(Des), 2,2′-Diaminopimelic acid (Dpm), 2,3-Diaminopropionic acid (Dpr),N-Ethylglycine (EtGly), N-Ethylasparagine (EtAsn), Hydroxylysine (Hyl),allo-Hydroxylysine (AHyl), 3-Hydroxyproline (3Hyp), 4-Hydroxyproline(4Hyp), Isodesmosine (Ide), allo-Isoleucine (Alle), N-Methylglycine(MeGly, sarcosine), N-Methylisoleucine (Melle), 6-N-Methyllysine(MeLys), N-Methylvaline (MeVal), Norvaline (Nva), Norleucine (Nle), andOrnithine (Orn).

Other examples of modified and unusual amino acids are describedgenerally in Synthetic Peptides: A User's Guide, Second Edition, April2002, Edited Gregory A. Grant, Oxford University Press ; Hruby V J,Al-obeidi F and Kazmierski W: Biochem J 268:249-262, 1990; and TonioloC: Int J. Peptide Protein Res 35:287-300, 1990; the teachings of all ofwhich are expressly incorporated herein by reference.

In certain example embodiments, the amino acid sequence of the peptidesis sequential, without any modified and unusual amino acids interruptingthe sequence of D- or L-amino acids. In other embodiments, the sequencemay include one or more modified and unusual amino acids as noted above.For example, the sequence of the peptides may be interrupted by one ormore modified and unusual amino acids. Accordingly, provided arepseudopeptides and peptidomimetics, including structures that have anon-peptidic backbone that specifically bind amyloids. In certainexample embodiments, the amyloid-reactive peptides and fusion peptidesinclude dimers or multimers of peptides that have enhanced affinity foramyloids as compared to their monomers.

In certain example embodiments, the amyloid-reactive peptides and fusionpeptides may be rich in positively charged amino acids. For example, theamyloid-reactive peptides and fusion peptides may include at least about15% positively charged amino acids such as arginine or lysine. In otherexample embodiments, the amyloid-reactive peptides and fusion peptidesmay include from about 15% to about 50%, about 20% to about 45%, about25% to about 40%, or about 30% to about 35% positively charged aminoacids, such as arginine or lysine.

In certain example embodiments, particular amyloid-reactive peptides andfusion peptides include one or more of the amino acid sequences setforth as SEQ ID NOS:1-17, as shown in Table 2 (below).

TABLE 2 Example Amyloid-Reactive Peptide Sequences PEPTIDEPRIMARY SEQUENCE: SEQ ID NO P5 KAQKA QAKQA KQAQK SEQ ID NO: 1AQKAQ AKQAK Q p5R RAQRA QARQA RQAQR SEQ ID NO: 2 AQRAQ ARQAR Q p5GGAQGA QAGQA GQAQG SEQ ID NO: 3 AQGAQ AGQAG Q P8 KAKAK AKAKA KAKAKSEQ ID NO: 4 P9 KAQAK AQAKA QAKAQ SEQ ID NO: 5 AKAQA KAQAK AQAK p19KAQQA QAKQA QQAQK SEQ ID NO: 6 AQQAQ AKQAQ Q p20 QAQKA QAQQA KQAQQSEQ ID NO: 7 AQKAQ AQQAK Q p31 KAQKA QAKQA KQAQK SEQ ID NO: 8AQKAQ AKQAK Q p37 KTVKT VTKVT KVTVK SEQ ID NO: 9 TVKTV TKVTK V p39[KAQKA QAKQA KQAQK SEQ ID NO: 10 AQKAQ AKQAK Q]_(D) p42V[Y]_(D)KVK TKVKT KVKTK SEQ ID NO: 11 VKT p43 [AQA]_(D)YS KAQKA QAKQASEQ ID NO: 12 KQAQK AQKAQ AKQAK Q p44 [AQA]_(D)YA QARQA RQAQRSEQ ID NO: 13 AQRAQ ARQAR Q p48 AQA[YS KAQKA QAKQA SEQ ID NO: 14KQAQK AQKAQ AKQAK Q]_(D) p50 AQAYS KAQKA QAKQA KQAQK SEQ ID NO: 15AQKAQ AKQAK Q p58 AQA[Y]_(D)S KAQKA QAKQA SEQ ID NO: 16KQAQK AQKAQ AKQAK Q p5 + 14 KAQKA QAKQA KQAQK AQKAQ SEQ ID NO: 17AKQAK QAQKA QKAQA KQAKQ Where D = the “D form” enantiomer.

In certain example embodiments, the amyloid-reactive peptides and fusionpeptides include a peptides that are at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95% identical to one or more of the sequences set forth as SEQ IDNOS:1-17. In certain example embodiments, the amyloid-reactive peptidesand fusion peptides may include a functional leader sequence fused tothe N-terminus or C-terminus of the peptide. For example, one or more ofthe sequences shown in Table 2 may include GGGYS-(SEQ ID NO:24) orCGGYS-(SEQ ID NO:25) sequences that are fused to the N-terminus end ofthe peptide. The leader sequence, for example, may be a cell-penetratingsequence.

In certain example embodiments, the amyloid-reactive peptides aremodified to include an epitope to a known antibody. That is, an epitopeof a known antibody or functional fragment thereof is attached to one ormore of the amyloid-reactive peptides described herein to form anamyloid-reactive fusion peptide. In accordance with the methodsdescribed herein, when the amyloid-reactive fusion peptide including theepitope comes in to contact with the antibody to which the epitope isreactive, the antibody binds the epitope (and hence indirectly binds theamyloid-reactive peptide via the epitope). As such, the antibody epitopecan be any antibody epitope, for example, that results in binding of theepitope to the antibody to which the epitope is reactive. In certainexample embodiments, any of the amyloid-reactive peptides identified inTable 2 (SEQ ID NOS:1-17) may be fused to such an epitope to form anamyloid-reactive fusion peptide.

In certain example embodiments, the epitope of the amyloid-reactivefusion peptide is added to the extreme N- or C-terminus of theamyloid-reactive peptide, since the ends of the proteins are more likelyto be accessible to the antibody and since the addition of the epitopeto the end is less likely to affect the function of the protein.Additionally or alternatively, addition of the epitope to an internalsite may be used, such as when the ends of the fusion peptide areimportant for the peptide's function or when processing is taking placeat these ends. In certain example embodiments, the amyloid-reactivefusion peptide may include a linker sequence to fuse the epitope to theamyloid-reactive peptide. The linker sequence may be any sequence knownin the art that, when used to form the fusion peptide, does notinterfere with the function of the peptide. In certain exampleembodiments, the linker has the following sequence: “SVTVVT” (SEQ ID NO:21).

In certain example embodiments, the epitope of the fusion peptide is anepitope of an antibody that binds to amyloids (i.e., the epitope is anepitope of an amyloid-reactive antibody). For example, the epitope is anepitope of an antibody that binds AA, AL, AH, ATTR, Aβ2M, ALect2, Wildtype, TTR, AApoAI, AApoAII, AGel, ALys, ALect2, Afib, ACys, ACal,AMedin, AIAPP, APro, Alns, APrP, Aβ amyloids, or any other amyloid. Insuch embodiments, the amyloid-reactive antibodies may bind both (1) theamyloid-reactive peptides including the epitope as well as (2) theamyloid type(s) to which the antibody is directed, as described herein.In certain example embodiments, the epitope may be a His-tag, Myc-tag,or other tag known in the art.

In certain example embodiments, the epitope is one that binds to the11-1F4 antibody or functional fragments thereof, the 11-1F4 antibodybeing described in U.S. Pat. No. 8,105,594 and in O'Nuallain et al.,Biochemistry, 2007, 46 (5), 1240-1247 (both of which are expresslyincorporated herein by reference in their entirety). For example, theLen(1-16) peptide, which is a known binding motif of theamyloid-reactive monoclonal antibody 11-1F4, may be used as a basis forthe epitope. In such example embodiments, the monoclonal antibody 11-1F4binds to the Len(1-16)-based peptide-epitope (“peptope”) fusion via theepitope amino acid sequence rather than to the amyloid-reactive peptidedirectly, as described herein. For example, any of the amyloid-reactivepeptides in Table 2 may be fused to the Len(1-16)-based sequence“DIVMTQSPDS LAVSLG” (SEQ ID NO:22) to form an amyloid-reactive fusionpeptide as described herein. As an example, the amyloid-reactive fusionpeptide may include the amyloid-reactive peptide of SEQ ID NO:17 (p5+14)fused to the Len(1-16)-based sequence set forth in SEQ ID NO:22.

In certain example embodiments, an amyloid-reactive peptide is fused toan 11-1F4 antibody epitope having the following 12-mer epitope sequence:“KHYAAFPENLLI” (SEQ ID NO:23). In certain example embodiments, theKHYAAFPENLLI epitope sequence is fused to any of the amyloid-reactivepeptides in Table 2 to form the amyloid-reactive fusion peptide. As anexample, the amyloid-reactive peptide having the sequence set forth asSEQ ID NO:17 (p5+14) is fused to the KHYAAFPENLLI sequence to form anamyloid-reactive fusion peptide. In such example embodiments, the12-mer-epitope sequence may be indirectly fused to the peptide, such asto the C-terminus of the peptide, via a linker sequence as describedherein. For example, the linker sequence may be SVTVVT (SEQ ID NO: 21).In certain example embodiments, an amyloid-reactive fusion peptideincluding the 11-1F4 antibody 12-mer epitope fused to the p5+14 peptidehas the following amino acid sequence (SEQ ID NO: 18 or “p66”), with theunderlined portion being an 11-1F4 reactive 12-mer epitope and SVTVVTbeing the linker sequence:

KAQKA QAKQA KQAQK AQKAQ AKQAK QAQKA QKAQA KQAKQ SVTVVT KHYAAFPENLLI

In certain example embodiments, the amyloid-reactive fusion peptide hasthe following sequence (SEQ ID NO:19), where X is an amino acid of alinker sequence and the underlined portion is the 11-1F4 12-mer epitope:

KAQKA QAKQA KQAQK AQKAQ AKQAK QAQKA QKAQA KQAKQ-XXXXXX-KHYAAFPENLLIIn certain example embodiments, an amyloid-reactive fusion peptideincluding the 11-1F4 antibody 12-mer epitope has the following aminoacid sequence (SEQ ID NO:20), with the underlined portion being the11-1F4 reactive epitope, X being an amino acid of a linker sequence, and“n” being the number of linker amino acids:

KAQKA QAKQA KQAQK AQKAQ AKQAK QAQKA QKAQA KQAKQ-[X]_(n)-KHYAAFPENLLI

For example, “n” may equal any number of amino acids, so long as thefunction of the amyloid-reactive peptide and the epitope is preserved.For example, “n” may equal about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 amino acids.

In certain example embodiments, the amyloid-reactive fusion peptideincluding a 11-1F4 antibody 12-mer epitope is at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% identical to any of the sequences set forth as SEQID NOS: 18-20.

In certain example embodiments, the epitope may include an epitope orfunctional fragment thereof of monoclonal antibodies 2A4, 7D8, and 8G9.These antibodies, for example, are amyloid-reactive antibodies that bindto specific amyloid fibrils. See J. S. Wall, et al., AL Amyloid Imagingand Therapy with a Monoclonal Antibody to a Cryptic Epitope on AmyloidFibrils, PLoS ONE 7(12):e52686 (2012); J.S. Wall et al., Generation andCharacterization of anti-AA Amyloid-Specific Monoclonal Antibodies;Frontiers of Immunology doi:10.3389/fimmu.2011.00032 (2011) (both ofwhich are expressly incorporated herein by reference in their entirety).As such, epitopes or functional fragments thereof to these antibodies,for example, may be fused to the amyloid-reactive peptides describedherein to create fusion peptides having epitopes to one or more of the2A4, 7D8, or 8G9 antibodies. Hence, in certain example embodiments, theepitope described herein includes a 2A4, 7D8, or 8G9 antibody-bindingmotif With the epitope or functional fragment thereof of antibodies 2A4,7D8, or 8G9, such fusion peptides can target one or more of the 2A4,7D8, or 8G9 antibodies to a variety of amyloid types for clearance asdescribed herein.

In certain example embodiments, any of the amyloid-reactive peptides andfusion peptides described herein may include a functional leadersequence fused to the N-terminus end of the peptide. For example, one ormore of the sequences shown in Table 2 may include GGGYS-(SEQ ID NO:24)or CGGYS-(SEQ ID NO:25) sequences that are fused to the N-terminus endof the peptide isolated.

The amyloid-reactive peptides and amyloid-reactive fusion peptideshaving an antibody epitope described herein may be made by any techniqueknown to those of skill in the art, including chemical synthesis orrecombinant means using standard molecular biological techniques. Thepeptides may be synthesized in solution or on a solid support inaccordance with conventional techniques. Various automatic synthesizersare commercially available and can be used in accordance with knownprotocols. (See, for example, Stewart and Young, Solid Phase PeptideSynthesis, 2d ed. Pierce Chemical Co., 1984; Tam et al., J. Am. Chem.Soc., 105:6442, 1983; Merrifield, Science, 232: 341-347, 1986; andBarany and Merrifield, The Peptides, Gross and Meienhofer, eds.,Academic Press, New York, pp. 1-284, 1979, each of which is expresslyincorporated herein by reference in its entirety).

Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes an amyloid-reactive peptide asdescribed herein is inserted into an expression vector, transformed ortransfected into an appropriate host cell, cultivated under conditionssuitable for expression, and isolating the peptide.

In certain example embodiments, the amyloid-reactive peptides and fusionpeptides may be obtained by isolation or purification. Proteinpurification techniques involve, at one level, the homogenization andcrude fractionation of cells, tissue, or organs to peptide andnon-peptide fractions. Other protein purification techniques include,for example, precipitation with ammonium sulfate, polyethylene glycol(PEG), antibodies and the like, or by heat denaturation, followed by:centrifugation; chromatography steps such as ion exchange, gelfiltration, reverse phase, hydroxylapatite and affinity chromatography;isoelectric focusing; gel electrophoresis, for example polyacrylamidegel electrophoresis; and combinations of these and other techniques.

Various chromatographic techniques include but are not limited toion-exchange chromatography, gel exclusion chromatography, affinitychromatography, immuno-affinity chromatography, and reverse phasechromatography. A particularly efficient method of purifying peptides isfast performance liquid chromatography (FPLC) or even high performanceliquid chromatography (HPLC).

The order of conducting the various purification steps may be changed,for example, or certain steps may be omitted, and still result in asuitable method for the preparation of a substantially purified peptide.

The peptides may be a part of a polypeptide or protein and may beproduced by biochemical or enzymatic fragmentation of the polypeptide orprotein. Accordingly, the peptides of the present invention may be (a)produced by chemical synthesis, (b) produced by recombinant DNAtechnology, (c) produced by biochemical or enzymatic fragmentation oflarger molecules, (d) produced by methods resulting from a combinationof methods a through d listed above, or (e) produced by any other meansfor producing peptides known to those of skill in the art.

During chemical synthesis, the amyloid-reactive peptides may be modifiedat the N- or C-terminus, thereby providing for improved stability andformulation, resistance to protease degradation, and the like. Examplesof modifications of amino acids include pegylation, acetylation,alkylation, formylation, amidation. Moreover, various amino acids thatdo not naturally occur along the chain may be introduced to improve thestability of the peptides.

In certain example embodiments, also provided are nucleic acid moleculesencoding the amyloid-reactive peptides and fusion peptides describedherein. For example, the nucleic acid molecules include a nucleic acidsequence encoding an amino acid sequence at least 95% identical to theamino acids set forth as any one of SEQ ID NOS: 1-20, such as at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to the amino acids set forth as any one of SEQ ID NOS: 1-20.In the context of the compositions and methods described herein, anucleic acid sequence that encodes at least one amyloid-reactive peptideor fusion peptide, such as described herein, is incorporated into avector capable of expression in a host cell (for example an adenoviralvector), using established molecular biology procedures. For examplenucleic acids, such as cDNAs, that encode at least one amyloid-reactivepeptide or fusion peptide can be manipulated with standard proceduressuch as restriction enzyme digestion, fill-in with DNA polymerase,deletion by exonuclease, extension by terminal deoxynucleotidetransferase, ligation of synthetic or cloned DNA sequences,site-directed sequence-alteration via single-stranded bacteriophageintermediate or with the use of specific oligonucleotides in combinationwith PCR or other in vitro amplification.

Example procedures sufficient to guide one of ordinary skill in the artthrough the production of vector capable of expression in a host cellthat includes a polynucleotide sequence that encodes at least oneamyloid-reactive peptide or fusion as described herein can be found forexample in Sambrook et ah, Molecular Cloning: A Laboratory Manual, 2ded., Cold Spring Harbor Laboratory Press, 1989; Sambrook et ah,Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring HarborPress, 2001; Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates, 1992 (and Supplements to 2003); andAusubel et al, Short Protocols in Molecular Biology: A Compendium ofMethods from Current Protocols in Molecular Biology, 4th ed., Wiley &Sons, 1999 (each of which are hereby expressly incorporated in theirentirety).

Typically, a polynucleotide sequence encoding at least oneamyloid-reactive peptide or fusion peptide is operably linked totranscriptional control sequences including, for example a promoter anda polyadenylation signal. A promoter is a polynucleotide sequencerecognized by the transcriptional machinery of the host cell (orintroduced synthetic machinery) that is involved in the initiation oftranscription. A polyadenylation signal is a polynucleotide sequencethat directs the addition of a series of nucleotides on the end of themRNA transcript for proper processing and trafficking of the transcriptout of the nucleus into the cytoplasm for translation.

Exemplary promoters include viral promoters, such as cytomegalovirusimmediate early gene promoter (“CMV”), herpes simplex virus thymidinekinase (“tk”), SV40 early transcription unit, polyoma, retroviruses,papilloma virus, hepatitis B virus, and human and simianimmunodeficiency viruses. Other promoters are isolated from mammaliangenes, including the immunoglobulin heavy chain, immunoglobulin lightchain, T-cell receptor, HLA DQ α and DQ β, β-interferon, interleukin-2,interleukin-2 receptor, MHC class II, HLA-DRα, β-actin, muscle creatinekinase, prealbumin (transthyretin), elastase I, metallothionein,collagenase, albumin, fetoprotein, β-globin, c-fos, c-HA-ras, insulin,neural cell adhesion molecule (NCAM), al -antitrypsin, H2B (TH2B)histone, type I collagen, glucose-regulated proteins (GRP94 and GRP78),rat growth hormone, human serum amyloid A (SAA), troponin I (TNI),platelet-derived growth factor, and dystrophin, dendritic cell-specificpromoters, such as CD1 Ic, macrophage-specific promoters, such as CD68,Langerhans cell-specific promoters, such as Langerin, and promotersspecific for keratinocytes, and epithelial cells of the skin and lung.

The promoter can be either inducible or constitutive. An induciblepromoter is a promoter that is inactive or exhibits low activity exceptin the presence of an inducer substance. Examples of inducible promotersinclude, but are not limited to, MT II, MMTV, collagenase, stromelysin,SV40, murine MX gene, α-2- macroglobulin, MHC class I gene h-2kb, HSP70,proliferin, tumor necrosis factor, or thyroid stimulating hormone genepromoter.

Typically, the promoter is a constitutive promoter that results in highlevels of transcription upon introduction into a host cell in theabsence of additional factors. Optionally, the transcription controlsequences include one or more enhancer elements, which are bindingrecognition sites for one or more transcription factors that increasetranscription above that observed for the minimal promoter alone. It maybe desirable to include a polyadenylation signal to effect propertermination and polyadenylation of the gene transcript. Examplepolyadenylation signals have been isolated from bovine growth hormone,SV40 and the herpes simplex virus thymidine kinase genes. Any of theseor other polyadenylation signals can be utilized in the context of theadenovirus vectors described herein.

Methods of generating fusion peptides, such as the amyloid-reactivefusion peptides described herein, are also well known to those of skillin the art. Such proteins can be produced, for example, by chemicalattachment using bifunctional cross-linking reagents, by de novosynthesis of the complete fusion peptide, or by attachment of a DNAsequence encoding the pre-targeting peptide to a DNA sequence encodingthe second peptide or protein, followed by expression of the intactpeptide or fusion peptide.

Host cells for expressing the amyloid-reactive peptides and fusionpeptides described herein include prokaryotes or eukaryotes. Suitableprokaryote hosts include bacterial host cells such as E. Coli. Variousstrains of E. coli include but are not limited to HB101, DHS, DH10, andMC1061. Suitable eukaryote hosts include yeasts and mammalian cells.Examples include but are not limited to Saccharomyces (e.g. S.cerevisiae); 293 (human embryonic kidney) (ATCC CRL-1573); 293F(Invitrogen, Carlsbad Calif.); 293T and variant 293T/17 (293tsA1609neoand variant ATCC CRL-11268) (human embryonic kidney transformed by SV40T antigen); COS-1 and COS 7 (monkey kidney CVI line transformed bySV40)(ATCC CRL1651); BHK (baby hamster kidney cells) (ATCC CRL10); CHO(Chinese hamster ovary cells); mouse Sertoli cells; CVI (monkey kidneycells) (ATCC CCL70); VERO76 (African green monkey kidney cells) (ATCCCRL1587); HeLa (human cervical carcinoma cells) (ATCC CCL2); MDCK(canine kidney cells) (ATCC CCL34); BRL3A (buffalo rat liver cells)(ATCC CRL1442); W138 (human lung cells) (ATCC CCL75); HepG2 (human livercells) (HB8065); and MMT 060652 (mouse mammary tumor) (ATCC CCL51).

Further exemplary mammalian host cells include primate cell lines androdent cell lines, including transformed cell lines. Normal diploidcells, cell strains derived from in vitro culture of primary tissue, aswell as primary explants, are also suitable. Candidate cells may begenotypically deficient in the selection gene, or may contain adominantly acting selection gene. Other suitable mammalian cell linesinclude but are not limited to, HeLa, mouse L-929 cells, 3T3 linesderived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines.

The amyloid-reactive peptides and fusion peptides described herein maybe produced by transforming or transfecting host cells with nucleicacids encoding the amyloid-reactive peptides and fusion peptides.Methods for transforming and transfecting host cells with nucleic acidsare well known and routinely performed. The nucleic acid sequencesencoding the amyloid-reactive peptides and fusion peptides describedherein also may be introduced into cultured mammalian cells by, forexample, calcium phosphate-mediated transfection (Wigler et al., Cell14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981;Graham and Van der Eb, Virology 52: 456, 1973). Other techniques forintroducing cloned DNA sequences into mammalian cells, such aselectroporation (Neumann et al., EMBO J. 1: 841-845, 1982), orlipofection may also be used. In order to identify cells that haveintegrated the cloned DNA, a selectable marker is generally introducedinto the cells along with the gene or cDNA of interest. Examples ofselectable markers for use in cultured mammalian cells include genesthat confer resistance to drugs, such as neomycin, hygromycin, andmethotrexate. The selectable marker may be an amplifiable selectablemarker, for example, the DHFR gene and the DHFRr. Selectable markers arereviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers,Stoneham, Mass.) and the choice of selectable markers is well within thelevel of ordinary skill in the art.

Antibodies to Amyloid-Reactive Peptides and Fusion Peptides

As provided herein, any antibodies that bind amyloid-reactive peptidesand fusion peptides can be used within the scope of the methods andcompositions described herein. More particularly, the variousamyloid-reactive peptides and fusion peptides described herein bind toamyloids. Hence, binding of an antibody to one or more of theamyloid-reactive peptides and fusion peptides results in targeting ofthe antibody to the amyloid. As such, an antibody that binds to theamyloid-reactive peptides and fusion peptides described herein may beused within the scope of the present disclosure to target antibodies toamyloid deposits.

In certain example embodiments, the antibodies specifically bind any oneof the amyloid-reactive peptides having the sequence set forth in SEQ IDNOS. 1-17 in Table 2. In certain example embodiments, the antibodiesbind to one or more functional, peptide fragments of theamyloid-reactive peptides that are at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% identical to one or more of the sequences set forth as SEQ IDNOS:1-17 in Table 2.

In certain example embodiments, antibodies to the amyloid-reactivepeptides are generated by immunizing a suitable host with peptide p43(SEQ ID NO: 12) and AA amyloid containing material. In such embodiments,the generated antibodies may be reactive to amyloid-reactive peptidesp5, p9, p31, p43, p44, p50, p58, and p5+14 (see Table 3 (below) forantibody reactivity and Table 2 (above) for corresponding sequenceidentification number designations of the peptides). In certain exampleembodiments, provided are cell lines producing the antibodies providedin Table 3 below. Also provided are sub-clones and variant clones ofsuch cell lines, which still produce an antibody with amyloid-reactiveprotein binding properties of as described herein.

The antibodies described herein may be human, humanized, or chimericantibodies. In certain example embodiments, the antibodies may be human,humanized, or chimeric antibodies that specifically bind to any one ofthe amyloid-reactive peptides having the sequence set forth as SEQ IDNOS. 1-17 or fragments thereof. For example, the antibodies may behuman, humanized, or chimeric antibodies that specifically bindfunctional peptide fragments of the amyloid-reactive peptides that areat least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% identical to one or more of thesequences set forth as SEQ ID NOS. 1-17. In certain example embodiments,the antibodies may be human, humanized, or chimeric antibodies that bindan epitope of the amyloid-reactive fusion peptides described herein. Forexample, the antibody may be a humanized 11-1F4 antibody or functionalfragment thereof that binds the Len(1-16) epitope of the 11-1F4 12-merepitope described herein.

In certain example embodiments, the human antibody is of human isotypeIgG1, IgG2, IgG3 or IgG4. In certain example embodiments, the humanizedantibody is of human isotype IgG1, IgG2, IgG3 or IgG4. In certainexample embodiments, the chimeric antibody is of human isotype IgG1,IgG2, IgG3 or IgG4. In certain example embodiments, the antibody is amouse antibody or rabbit antibody. In certain example embodiments, theantibody is a polyclonal antibody. In certain example embodiments, theantibody is a monoclonal antibody. For example, the antibody is amonoclonal antibody that recognizes a specific epitope on or attached tothe amyloid-reactive peptide.

In certain example embodiments, the antibodies are amyloid-reactiveantibodies. For example, the antibody can be a monoclonal antibody (or“mAB”) that recognizes an epitope that is common to both anamyloid-reactive fusion peptide and a specific amyloid. Example amyloidsto which the amyloid-reactive antibodies may bind include but are notlimited to one or more of AA, AL, AH, ATTR, Aβ2M, ALect2, Wild type,TTR, AApoAI, AApoAII, AGel, ALys, ALect2, Afib, ACys, ACal, AMedin,AIAPP, APro, AIns, APrP, or AP amyloids. In such example embodiments,the amyloid-reactive peptide is fused to an epitope recognized by theantibody as described herein. Hence, the amyloid-reactive antibodyrecognizes the fused epitope of the amyloid-reactive fusion peptide. Theamyloid-reactive antibody also recognizes an amyloid directly, such asvia a common epitope of the amyloid from which the epitope is derived.

In certain example embodiments, the antibody is an 11-1F4 antibody orfunctional fragments thereof that binds an amyloid-reactive fusionpeptide. The 11-1F4 antibody, for example, has been shown to bind ALamyloid in patients with AL. Yet not all subjects are immunoreactive to11-1F4 and this mAb does not bind ATTR or AA amyloid in vivo. Therefore,to advantageously enhance the utility of the 11-1F4 antibody, a bindingmotif of the 11-1F4 antibody may be fused to an amyloid-reactive peptideas described herein.

For example, an 11-1F4 binding motif may be fused to one or more panamyloid-reactive peptides as described herein to result in anamyloid-reactive fusion peptide. The 11-1F4 antibody, which when usedalone has the disadvantages noted above, can then advantageously be usedas a single antibody to target multiple amyloid types via binding to thepan amyloid-reactive fusion peptide. In other words, the use of 11-1F4or fragments thereof can be expanded beyond interaction with a fewamyloid types and can be used in subjects otherwise not immunoreactive.

In certain example embodiments, the 11-1F4 antibodies may bind aLen(1-16)-based epitope that is fused to the amyloid-reactive peptide ofthe amyloid-reactive fusion peptide. Additionally or alternatively, the11-1F4 antibody may bind to an amyloid-reactive fusion peptide havingthe sequence set forth as SEQ ID NOS:18, which includes an 11-1F4 12-merbinding motif and linker region. Additionally or alternatively, the11-1F4 antibody may bind to an amyloid-reactive fusion peptide havingthe sequence set forth as any one of SEQ ID NOS:19-20, which includes an11-1F4 12-mer binding motif and a variable linker region.

In accordance with the methods described herein, use of anamyloid-reactive antibody that binds to a pan amyloid-reactive fusionpeptide has the advantage of (1) using a single antibody to target aspecific amyloid directly and (2) using the same, single antibody totarget a vast array of other amyloid types via the amyloid-reactivepeptide (when the antibody alone may not otherwise bind the variety ofamyloid types). For example, with the present disclosure, use of the11-1F4 antibody is greatly expanded to treat a variety of amyloid-baseddiseases via targeting of the 11-1F4 antibody to multiple amyloid typesvia an amyloid-reactive fusion peptide that includes an 11-1F4 bindingmotif.

In certain example embodiments, functional fragments of the antibodiesdescribed herein may be used in accordance with the methods andcompositions provided herein. For example, fragments comprising only aportion of the primary antibody structure may be produced wherein thefragment substantially retains the immunoreactive properties theantibody. Such fragments include, for example, fragments produced byproteolytic cleavage of intact antibodies by methods well known in theart, or fragments produced by inserting stop codons at the desiredlocations in the nucleotide sequence using site-directed mutagenesis.For example, a stop codon can be inserted after CH1 to produce Fabfragments or after the hinge region to produce F(ab′)2fragments. Singlechain antibodies and fusion proteins that include at least animmunoreactive fragment are also included within the scope of theinvention. In certain example embodiments, the antibody or fragmentthereof may be directly or indirectly attached to effector moietieshaving therapeutic activity. Suitable effector moieties includecytokines, cytotoxins, radionuclides, drugs, immunomodulators,therapeutic enzymes, anti-proliferative agents, etc. Methods forattaching antibodies to such effectors are well known in the art.

Antibodies to the amyloid-reactive peptides and fusion peptides providedherein can be prepared using any method. For example, any substantiallypure amyloid-reactive peptide or fragment thereof can be used as animmunogen to elicit an immune response in an animal such that specificantibodies are produced. For example, any of amyloid-reactive peptidesor fragments thereof having the sequence set forth as SEQ ID NOS: 1-17may be used as an immunizing antigen to generate antibodies to theamyloid-reactive peptides.

In certain example embodiments, any of amyloid-reactive peptidesdescribed herein or fragments thereof may be combined with murine AAamyloid-containing material (amyloid-enhancing factor or “AEF”). Thecomplex of amyloid-reactive peptides or fragments thereof with the AEFcan then be used as the immunogen. For example, peptide p43 (SEQ ID NO:12) may be mixed with AEF. Mice may then be immunized with a suspensionof complexed AEF/p43 to generate the antibodies to the amyloid-reactivepeptide (see Table 3 herein).

Additionally or alternatively, the immunogen used to immunize an animalcan be chemically synthesized or derived from translated cDNA. Further,the immunogen can be conjugated to a carrier polypeptide, if desired.Commonly used carriers that are chemically coupled to an immunizingpolypeptide include, without limitation, keyhole limpet hemocyanin(KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.

The preparation of polyclonal antibodies is well known to those skilledin the art. See, e.g., Green et al, Production of Polyclonal Antisera,in Immunochemical Protocols (Manson, ed.), pages 15 (Humana Press 1992)and Coligan et al, Production of Polyclonal Antisera in Rabbits, Rats,Mice and Hamsters, in Current Protocols in Immunology, section 2.4.1(1992). In addition, those of skill in the art will know of varioustechniques common in the immunology arts for purification andconcentration of polyclonal antibodies, as well as monoclonal antibodies(Coligan, et al., Unit 9, Current Protocols in Immunology, WileyInterscience, 1994).

The preparation of monoclonal antibodies is also well known to thoseskilled in the art. See, e.g., Kohler & Milstein, Nature 256:495 (1975);Coligan et al, sections 2.5.1 2.6.7; and Harlow et al, Antibodies: ALaboratory Manual, page 726 (Cold Spring Harbor Pub. 1988). Briefly,monoclonal antibodies can be obtained by injecting mice with acomposition comprising an antigen, such as one of the amyloid-reactivepeptides described herein, verifying the presence of antibody productionby analyzing a serum sample, removing the spleen to obtain Blymphocytes, fusing the B lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures. Monoclonal antibodies can be isolated and purifiedfrom hybridoma cultures by a variety of well-established techniques.Such isolation techniques include affinity chromatography with Protein ASepharose, size exclusion chromatography, and ion exchangechromatography. See, e.g., Coligan et al, sections 2.7.1 2.7.12 andsections 2.9.1 2.9.3; Barnes et al, Purification of Immunoglobulin G(IgG), in Methods In Molecular Biology, VOL. 10, pages 79 104 (HumanaPress 1992).

In addition, methods of in vitro and in vivo multiplication ofmonoclonal antibodies is well known to those skilled in the art.Multiplication in vitro can be carried out in suitable culture mediasuch as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionallyreplenished by mammalian serum such as fetal calf serum, or traceelements and growth sustaining supplements such as normal mouseperitoneal exudate cells, spleen cells, and bone marrow macrophages.Production in vitro provides relatively pure antibody preparations andallows scale up to yield large amounts of the desired antibodies. Largescale hybridoma cultivation can be carried out by homogenous suspensionculture in an airlift reactor, in a continuous stirrer reactor, or inimmobilized or entrapped cell culture. Multiplication in vivo may becarried out by injecting cell clones into mammals histocompatible withthe parent cells (e.g., osyngeneic mice) to cause growth of antibodyproducing tumors. In certain example embodiments, the animals are primedwith a hydrocarbon, especially oils such as pristane(tetramethylpentadecane) prior to injection. After one to three weeks,the desired monoclonal antibody is recovered from the body fluid of theanimal. In some cases, the antibodies provided herein can be made usingnon-human primates. General techniques for raising therapeuticallyuseful antibodies in baboons can be found, for example, in Goldenberg etal, International Patent Publication WO 91/11465 (1991) and Losman etal, Int. J. Cancer, 46:310 (1990).

In certain example embodiments, the antibodies can be humanizedmonoclonal antibodies. Humanized monoclonal antibodies can be producedby transferring mouse complementarity determining regions (CDRs) fromheavy and light variable chains of the mouse immunoglobulin into a humanvariable domain, and then substituting human residues in the frameworkregions of the murine counterparts. The use of antibody componentsderived from humanized monoclonal antibodies alleviates potentialproblems associated with the immunogenicity of murine constant regionswhen treating humans. General techniques for cloning murineimmunoglobulin variable domains are described, for example, by Orlandiet al., Proc. Natl Acad. Sci. USA, 86:3833 (1989). Techniques forproducing humanized monoclonal antibodies are described, for example, byJones et al., Nature, 321 :522 (1986); Riechmann et al, Nature, 332:323(1988); Verhoeyen et al, Science, 239:1534 (1988); Carter et al, Proc.Nat'l. Acad. Sci. USA, 89:4285 (1992); Sandhu, Crit. Rev. Biotech.,12:437 (1992); and Singer et al, J. Immunol, 150:2844 (1993).

Methods and Pharmacological Compositions

Therapeutic methods are provided for the treatment of amyloidosis,including amyloid diseases such as Alzheimer's disease, Huntington's orprion diseases, or peripheral such as seen in patients with light chain(AL) amyloidosis and type 2 diabetes. In certain example embodiments,the method includes selecting a subject with amyloidosis within whomamyloid deposits are to be cleared. The method also includesadministering an effective amount of one or more amyloid-reactivepeptides or amyloid-reactive fusion peptides to the subject. In certainexample embodiments, the subject may be administered one or more of thepeptides having the sequence set forth as SEQ ID NOS:1-20. The methodsfurther include administering an effective amount of one or moreantibodies described herein to the subject. Administration of theeffective amount of the amyloid-reactive peptides or fusion peptides—andthe antibodies described herein—results in clearance of amyloid depositin the subject.

Also provided herein are methods for clearing amyloid deposits. Forexample, an amyloid deposit is contacted with an amyloid-reactivepeptide or amyloid-reactive fusion peptide as described herein. Theamyloid-reactive peptide or amyloid-reactive fusion peptide is thencontacted with an antibody. The antibody binds the amyloid-reactivepeptide and targets the amyloid deposit for clearance. Contacting theamyloid-reactive peptide or fusion peptide with the antibody that bindsthe amyloid-reactive peptide or fusion peptide targets the amyloiddeposit for clearance.

In certain example embodiments, binding of the antibody or functionalfragment thereof to an epitope fused to the amyloid-reactive fusionpeptide results in increased clearance of the amyloid deposit from thesubject. For example, when the antibody is an amyloid-reactive antibody,administration of the antibody alone (without the amyloid-reactivepeptide) may result in some clearance of an amyloid deposit. However,when the amyloid-reactive antibody is administered following theadministration of the amyloid-reactive fusion peptide, increasedclearance is achieved. That is, the level of clearance may be greatervia the use of the amyloid-reactive fusion peptide versus use of theamyloid-reactive antibody alone. In certain example embodiments, anincrease in clearance may be observed in a subject. For example, thesubject may initially be provided with amyloid-reactive antibody alonewith limited improvement, i.e., little reduction in amyloid deposits.However, administration of the amyloid-reactive fusion peptide andamyloid-reactive antibody as described herein may result in greaterclearance of the amyloid deposits and hence improvement in the subject.

In certain example embodiments, the methods provided herein includeeliciting an immune response at the site of antibody binding to theantibody deposit via the amyloid-reactive peptide or fusion peptide. Forexample, administering an amyloid-reactive peptide or fusion peptide andantibody to a subject or contacting an amyloid deposit with theamyloid-reactive peptide or fusion peptide and antibody results inaccumulation of immune cells as the site of the deposit. The immunecells, for example, may be macrophages or other any other cells known orimplicated in an immune response that clear amyloid deposits.Advantageously, the methods and pharmaceutical compositions providedherein are able to target amyloid deposits for clearance while notaffecting healthy tissue.

In certain example embodiments, the antibody is administered to asubject or placed in to contact with the amyloid deposit after asufficient clearance period. For example, in a subject with amyloidosisthe antibody is administered after a sufficient clearance period thatallows unbound amyloid-reactive peptide to be cleared from the subject'ssystem. That is, the antibody is provided to the subject after asufficient time passes for the amyloid-reactive peptides or fusionpeptide to bind amyloids and for excess amyloid-reactive peptides orfusion peptides to be eliminated from the subject. Hence, in certainexample embodiments the antibody is administered at about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 24, 30, 36, 42, 48, 54, 60, 66, 72,78, 84, or 96 hours after administration of the amyloid-reactive peptideor fusion peptide.

In accordance with the methods described herein, FIG. 1 providesschematic drawings showing an example of targeting of antibodies toamyloids. As shown in FIG. 1A, the example amyloid-reactive peptide p5or p5+14 pre-targets AL, ATTR, and AA amyloids. An anti-p5 antibodypeptide then targets the p5 peptide. As shown in FIG. 1B, for example,an Al2-based epitope of the 11-1F4 antibody (the 11-1F4 12-mer describedherein) is fused to the C-terminus end of the p5 or p5+14 peptide. Withthe bound epitope, the amyloid-reactive peptide p5 or p5+14 pre-targetsAL, ATTR, and AA amyloids and then 11-1F4 antibody binds the epitope.Binding of the 11-1F4 antibody to the epitope thus targets the 11-1F4antibody to the p5 or p5+14 peptide and hence to the amyloid (FIG. 1B).

Without wishing to be bound by any particular theory, it is believedthat pre-targeting of the antibodies described herein to amyloiddeposits via amyloid reactive peptides elicits a host immune response tothe site of the amyloid deposit. The immune response, in turn, resultsin clearance of the amyloid deposits, for example, through processessuch as opsonization and phagocytosis. For example, anti-amyloidantibodies have been shown to clear injected amyloidomas in mice (U.S.Pat. No. 8,105,594). Further, an example immune response in the liver isillustrated in FIG. 19, where macrophages are shown to accumulate totargeted amyloids in mice in vivo. For example, mice injected withpeptide p66 and 11-1F4 antibody show induced macrophage infiltration atthe site of amyloids at 72 hours post treatment (See FIG. 19).

In certain example methods provided herein, a subject is administered aneffective amount of p66 peptide or functional fragment thereofThereafter, such as 24-48 hours later, the subject is administered aneffective amount of the 11-1F4 antibody or functional fragment thereofAdministration of the 11-1F4 antibody results in clearance of theamyloid deposits in the subject. In certain example embodiments providedherein, an amyloid deposit is contacted with p66 peptide or functionalfragment thereof Thereafter, the amyloid deposit is contacted with the11-1F4 antibody or functional fragment thereof Contacting the amyloiddeposit with the 11-1F4 antibody results in clearance of the amyloiddeposit. For example, administering the 11-1F4 antibody to thesubject—or contacting an amyloid deposit with the 11-1F4antibody—elicits an immune response at the site of the amyloid deposit,such as by eliciting macrophage or other immune cell accumulation to thesite of the amyloid deposit.

Also provided herein are pharmaceutical compositions for the treatmentof amyloid diseases, including pharmaceutical compositions that may beused in any of the methods provided herein. The purpose of apharmaceutical composition is to facilitate administration of a compoundor substance to the subject, such as the peptides and antibodiesdescribed herein. The pharmaceutical compositions include, for example,amyloid-reactive peptides or fusion peptides. The compositions alsoinclude antibodies as described herein. In certain example embodiments,a single pharmaceutical composition for administration to a subjectincludes both (1) amyloid-reactive peptides or fusion peptides and (2)antibodies as described herein, whereas in other embodiments theamyloid-reactive peptides or fusion peptides and antibodies foradministration to a subject are in separate pharmaceutical compositions.Such pharmaceutical compositions comprise an effective amount of theamyloid-reactive peptide (or amyloid-reactive fusion peptide) and theantibodies to treat amyloidosis in a subject, such as by clearingamyloid deposits in the subject.

In certain example embodiments, the pharmaceutical compositions willinclude an appropriate solid or liquid carrier, depending upon theparticular mode of administration chosen. The pharmaceuticallyacceptable carriers and excipients useful are conventional and known tothose skilled in the art. For instance, parenteral formulations usuallycomprise injectable fluids that are pharmaceutically and physiologicallyacceptable fluid vehicles such as water, physiological saline, otherbalanced salt solutions, aqueous dextrose, glycerol or the like.Excipients that can be included are, for instance, other proteins, suchas human serum albumin or plasma preparations. If desired, thepharmaceutical composition to be administered can also contain minoramounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate.

Water may be the preferred carrier when the pharmaceutical compositionis administered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. The composition can also optionally containminor amounts of wetting or emulsifying agents, or pH buffering agents.Such compositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The compositions can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oralformulations can include standard carriers such as pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, etc. Examples of these and othersuitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences (above).

In certain example embodiments, the compositions may also include asolubilizing agent and a local anesthetic such as lignocaine to easepain at the site of the injection. The ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampule indicating the quantity of active agent.Where the composition is to be administered by infusion, it can bedispensed with an infusion bottle containing sterile pharmaceuticalgrade water or saline. Where the composition is administered byinjection, an ampule of sterile water for injection or saline can beprovided so that the ingredients may be mixed prior to administration.

Administration of therapeutic compositions can be by any common route aslong as the target tissue is available via that route. This includesorthotopic, intradermal subcutaneous, intramuscular, intraperitoneal, orintravenous injection routes. Additionally or alternatively, the routemay be oral, nasal, ocular, buccal, or other mucosal or topicaladministration. Such pharmaceutical compositions are usuallyadministered as pharmaceutically acceptable compositions that includephysiologically acceptable carriers, buffers or other excipients, asdescribed herein.

The dosage form of the pharmaceutical composition will be determined bythe mode of administration chosen. For instance, in addition toinjectable fluids, topical, inhalation, oral and suppositoryformulations can be employed. Topical preparations can include eyedrops, ointments, sprays and the like. Inhalation preparations can beliquid (e.g., solutions or suspensions) and include mists, sprays andthe like. Oral formulations can be liquid (e.g., syrups, solutions orsuspensions), or solid (e.g., powders, pills, tablets, or capsules).Suppository preparations can also be solid, gel, or in a suspensionform. For solid compositions, conventional non-toxic solid carriers caninclude pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in the art.

An effective amount of the pharmaceutical composition is determinedbased on the intended goal, for example, such as clearance of amyloiddeposits. The appropriate dose will vary depending on thecharacteristics of the subject, for example, whether the subject is ahuman or non-human, the age, weight, and other health considerationspertaining to the condition or status of the subject, the mode, route ofadministration, and number of doses, and whether the pharmaceuticalcomposition includes nucleic acids or viruses. Generally, thepharmaceutical compositions described herein are administered for thepurpose treating amyloidosis, via clearance of the amyloid deposits. Theamount of active compound(s) administered will be dependent on thesubject being treated, the severity of the affliction, and the manner ofadministration, and is best left to the judgment of the prescribingclinician. Within these bounds, the formulation to be administered willcontain a quantity of the active component(s) in amounts effective toachieve the desired effect in the subject being treated. In certainexample embodiments, a unit dosage can be about 0.1 to about 10 mg persubject per day. Dosages from about 0.1 up to about 100 mg per subjectper day may be used, particularly if the agent is administered to asecluded site and not into the circulatory or lymph system, such as intoa body cavity, or into a lumen of an organ.

In certain example embodiments, the pharmaceutical compositions can bedelivered by way of a pump (see Langer, supra; Sefton, CRC Crit. RefBiomed. Eng. 14:201, 1987; Buchwald et al., Surgery 88:507, 1980; Saudeket al., N Engl. J. Med. 321:574, 1989) or by continuous subcutaneousinfusions, for example, using a mini-pump. An intravenous bag solutioncan also be employed. One factor in selecting an appropriate dose is theresult obtained, as measured by the methods disclosed here, as aredeemed appropriate by the practitioner. Other controlled release systemsare discussed in Langer (Science 249:1527-33, 1990).

In certain example embodiments, a pump is implanted (for example seeU.S. Pat. Nos. 6,436,091; 5,939,380; and 5,993,414). Implantable druginfusion devices are used to provide patients with a constant andlong-term dosage or infusion of a therapeutic agent, including thepharmaceutical compositions described herein. Such device can becategorized as either active or passive.

Active drug or programmable infusion devices feature a pump or ametering system to deliver the composition into the patient's system. Anexample of such an active infusion device currently available is theMedtronic SYNCHROMED™ programmable pump. Passive infusion devices, incontrast, do not feature a pump, but rather rely upon a pressurized drugreservoir to deliver the agent of interest. An example of such a deviceincludes the Medtronic ISOMED™.

In certain example embodiments, the pharmaceutical compositions aredelivered by sustained-release systems. Suitable examples ofsustained-release systems include suitable polymeric materials (such as,semi-permeable polymer matrices in the form of shaped articles, forexample films, or mirocapsules), suitable hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, andsparingly soluble derivatives (such as, for example, a sparingly solublesalt). Sustained-release compositions can be administered orally,parenterally, intracistemally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), or as an oral ornasal spray. Sustained-release matrices include polylactides (U.S. Pat.No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556, 1983,poly(2-hydroxyethyl methacrylate)); (Langer et al., J. Biomed. Mater.Res.15:167-277, 1981; Langer, Chem. Tech. 12:98-105, 1982, ethylenevinyl acetate (Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid(EP 133,988).

Polymers can be used for ion-controlled release. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, Accounts Chem. Res. 26:537, 1993). Forexample, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston etal., Pharm. Res. 9:425, 1992; and Pec, J. Parent. Sci. Tech. 44(2):58,1990). Alternatively, hydroxyapatite has been used as a microcarrier forcontrolled release of proteins (Ijntema et al., Int. J. Pharm. 112:215,1994).

In certain other example embodiments, liposomes are used for controlledrelease as well as drug targeting of the pharmaceutical compositionsdescribed herein (Betageri et al., Liposome Drug Delivery Systems,Technomic Publishing Co., Inc., Lancaster, Pa., 1993). Numerousadditional systems for controlled delivery of therapeutic proteins areknown (for example, U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837;U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No.4,837,028; U.S. Pat. No. 4,957,735; and U.S. Pat. No. 5,019,369; U.S.Pat. No. 5,055,303; U.S. Pat. No. 5,514,670; U.S. Pat. No. 5,413,797;U.S. Pat. No. 5,268,164; U.S. Pat. No. 5,004,697; U.S. Pat. No.4,902,505; U.S. Pat. No. 5,506,206; U.S. Pat. No. 5,271,961; U.S. Pat.No. 5,254,342; and U.S. Pat. No. 5,534,496).

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thesubject. In one embodiment, the dosage is administered once as a bolus,but in another embodiment can be applied periodically until atherapeutic result is achieved. Generally, the dose is sufficient totreat or ameliorate symptoms or signs of disease without producingunacceptable toxicity to the subject. Systemic or local administrationcan be utilized.

In certain example embodiments, provided is a kit. The kit, for example,typically includes one or more of the amyloid-reactive peptides orfusion peptides described herein or functional fragments thereof, suchas one or more of the amyloid-reactive peptides or fusion peptideshaving the sequence set forth as SEQ ID NOS:1-20 (or functionalfragments thereof). The kit also includes an antibody as describedherein or functional fragments thereof. The amyloid-reactive peptides orfusion peptides and antibodies of the kit, for example, may beformulated as described herein into one or more pharmaceuticalcompositions. The kit can include instructional materials disclosingmeans of use of the amyloid-reactive peptides or fusion peptides andantibodies The instructional materials may be written, in an electronicform (e.g. computer diskette or compact disk) or may be visual (e.g.video files). The kits may also include additional components tofacilitate the particular application for which the kit is designed,i.e., the treatment of amyloidosis.

EXAMPLES

The following examples further illustrate the invention but should notbe construed as in any way limiting its scope.

Example 1

Targeting of Antibodies to Amyloids with Amyloid-Reactive Peptides

Peptide Synthesis and Purification

Peptides were chemically synthesized and purified by high pressureliquid chromatography (HPLC [1100 series; Agilent]) by elution from areverse-phase C3 matrix in a linear gradient of 0-50% acetonitrile inwater with 0.05% trifluoroacetic acid. Peptide peaks were eluted fromthe column using a flow rate of 1 mL/min; 1- mL fractions werecollected, peak fractions were pooled, and the mass was determined by MSusing a single quadropole MS (Applied Biosystems). The purified peptideswere lyophilized as 5 mg aliquots and we re-suspended inphosphate-buffered saline (150 mM NaCl, pH7.2; PBS) before use. There-suspended peptides were stored at 4° C. until use.

Antibody Production

Five murine monoclonal antibodies were produced by using, as animmunogen, the peptide p43 mixed with murine AA amyloid-containingmaterial (amyloid-enhancing factor). More particularly, mice wereimmunized with a suspension of complexed AEF/p43 (peptide and AEF weremixed and the complexes washed by centrifugation at 16, 000×g beforeresuspending in sterile PBS). Mice received four injections of AEF/p43immunogen (50 μg—total mass in the complexes) prepared in PBS/Alumadjuvant. Following immnunization, the mice were euthanized and themouse splenocytes were isolated and fused with SP 2/0 myeloma cells bystandard PEG fusion, plated, and after 10 days in culture supernatantswere screened for immunoreactivity with immunogen by direct ELISA. Thewells of a 96-well microplate were coated (sequentially) with AEF andp43 peptide. Culture supernatant from fused clones was added to thewells and bound mouse antibodies were detected using horseradishperoxidase (HRP)-labeled anti-mouse IgG. ABTS was used as thecolorimetric substrate and the reactivity was measured using a platereader (Synergy HT) at 405 nm. Cells in wells with reactivesupernatants, based on the ELISA described above, were subcloned bylimiting dilution and subsequent clones re-screened by ELISA forreactivity with AEF/p43, as above. Supernatants from immunopositivesubclones were then re-tested for binding to peptide or AEF alone coatedonto the wells of a 96-well ELISA plate. Clones reactive with AEF alonewere not utilized further; all other clones (clones 4, 5, 8, 12, and 13)reacted with both p43/AEF complexes and p43 alone. Peptide p43-reactiveclones were propagated, isotyped (all were shown to be IgG1, kappa), andcryopreserved. For subsequent experiments (monoclonal antibodies) mAbswere purified from the subclone supernatants by protein A-affinitychromatography. In some cases, the purified mAbs were labeled withbiotin by covalent linkage, using standard procedures (Pierce). Peptidereactive antibodies were further characterized for reactivity on a panelof amyloid-reactive, and related peptides by europium-linkedimmunosorbent assay (EuLISA). The five antibody clones, i.e., clone 4,5, 8, 12, and 13 were then further examined as described below.

Peptide Reactivity of Subclones

The reactivity of each of the five purified and biotinylated mAbs withsynthetic peptides, related in structure to p43 (see Table 2, SEQ ID NO:12), was tested by EuLISA. More particularly, wells of a 96-wellmicroplate were coated with 200 ng of synthetic peptide by incubationovernight. The wells were blocked by using a solution of 1% (w/v) BSA inphosphate buffered saline (pH 7.2) before addition of purified,biotinylated mAbs (clone 4, 5, 8, 12, or 13) added at 100 ng/well.Detection of bound mAb was achieved by addition of europium-conjugatedstreptavidin followed by enhancement solution (Perkin Elmer). The timeresolved fluorescence was measured using a Victor 3 plate reader(Wallac, Perkin Elmer).

As shown in Tables 3 and 4, each of the clones was reactive withpeptides with the heptad amino acid repeat, Lys-X-X-Lys-X-X-X composedof L-amino acids, where X is Ala or Gln. Alteration of the spacing ofthe Lys residues or substitution of Lys for Arg, or use of D-amino acidsin the heptad resulted in loss or decrease of antibody binding.

TABLE 3 Antibody Clone Immunogen Isotype Peptide Reactivity 4-2 p43 +AEF IgG1κ p5⁺; p9^(+/−); p31⁺; p43⁺; p50⁺; p58⁺; p5 + 14⁺ 5-1 p43 + AEFIgG1κ p5⁺; p9^(+/−); p31⁺; p43⁺; p50⁺; p58⁺; p5 + 14⁺ 8-1 p43 + AEFIgG1κ p43⁺; p44^(+/−) 12-3  p43 + AEF IgG1κ p5⁺; p9^(+/−); p31⁺; p43⁺;p50⁺; p58⁺; p5 + 14⁺ 13-2  p43 + AEF IgG1κ p5⁺; p9^(+/−); p31⁺; p43⁺;p50⁺; p58⁺; p5 + 14⁺

TABLE 4 (p43-AEF) Monoclonal Antibody Clone Reactivity Table mAb Clone:Peptide: 4-2 5-1 8-1 12-3 13-2 p5 + + − + + p5R − − − − − p5G − − − − −p8 − − − − − p9 +/− +/− − +/− +/− p19 − − − − − p20 − − − − − p31 + +− + + p37 − − − − − p39 − − − − − p42 − − − − − p43 + + + + + p44 − −+/− − − p48 − − − − − p50 + + − + + p58 + + − + + p5 + 14 + + − + +

Amyloid Fibril Pre-targeting with p43 and p5+14 Peptides

The reactivity of each of the five mAbs to synthetic amyloid fibrils wastested by europium-linked immunosorbent assay (EuLISA). Moreparticularly, wells of a 96-well microplate were coated with 500 ng ofeither murine AA amyloid-associated amyloid extract (AEF) or syntheticlight chain-associated (AL) synthetic fibrils composed of the λ2,6variable domain (rVλ6Wil, aka WIL) by incubation overnight. The wellswere blocked by using a solution of 1% (w/v) BSA in phosphate bufferedsaline (pH 7.2) before addition of either peptide p43 (the immunogen) orpeptide p5+14 (100 ng/well). The wells were then washed and biotinylatedmAbs (clone 4, 5, 8, 12, or 13) added at 100 ng/well. Detection of boundmAb was achieved by addition of europium-conjugated streptavidinfollowed by enhancement solution (Perkin Elmer). The time resolvedfluorescence was measured using a Victor 3 plate reader (Wallac, PerkinElmer).

As shown in FIG. 2, all mAbs were reactive in the presence of fibrilscoated with peptide p43, the immunogen. However, only clones 4, 5, 12,and 13 bound amyloid fibrils in the presence of peptide p5+14. There wasno binding of the mAbs to the fibrils in the absence of pre-targetingpeptide. As shown in FIG. 3, similar data were obtained when thepeptides were used to pre-target murine AA amyloid extract (amyloidenhancing factor; AEF).

Capture of Pre-targeting Peptide

To determine whether mAb clones 4, 5, 12, and 13 clones are capable ofcapturing biotinylated peptide p5+14 from solution, we used a standardELISA assay. More particularly, 96-well ELISA microplates were coatedovernight with the indicated mAbs (500 ng/well), the wells were blockedwith PBS/BSA solution before addition of biotinylated p5+14 peptide(10Ong/well). Biotinylated peptides, p5R and p31G (aka p5G) were used asa negative control. Following a wash step, detection of captured peptidewas achieved by addition of europium-conjugated streptavidin asdescribed above.

As shown in FIG. 4, mAb clones 4, 5, 12, and 13 were shown capable ofcapturing biotinylated peptide p5+14 from solution when they wereadsorbed onto the wells of the microplate. In contrast, the biotinylatedforms of peptides p5R and p5G that are not reactive with any of theclones were not captured (FIG. 4). The mAb clone 8 does not bind peptidep5+14 when bound to rVλ6Wil fibrils, nor AA-AEF, and did not capturebiotinylated p5+14 in solution.

Binding to AA-AEF Amyloid Extract

In another assay, the ability of the mAb clones to bind AA-AEF amyloidextract was examined after being pre-incubated with theamyloid-targeting peptide p5+14 to form a complex. More particularly,ELISA wells were coated with AA amyloid-associated extract (AEF: 500ng/well overnight), The wells were blocked by using a solution of 1%(w/v) BSA in phosphate buffered saline (pH 7.2) before addition of asolution of peptide p5+14 and biotinylated mAb (4, 5, 8, 12, or 13) at1:2, 1:1, or 1:0.5 peptide:mAb molar ratio—pre-incubated for 90 min.After one hour incubation, the plates were washed and detection of boundmAb was achieved by addition of europium-conjugated streptavidinfollowed by enhancement solution, as described above. As shown in FIG.5, the binding was compared to standard pre-targeting with p5+14 beforeadding the mAb clones.

Pre-targeting Immunohistochemistry

The pre-targeting efficacy of peptide p5+14 in conjunction with mAbclones 4, 5, 12, or 13 was further evaluated using human ATTR-ladenformalin-fixed paraffin embedded tissue sections. More particularly, sixmicrometer-thick sections, cut from formalin-fixed, paraffin embeddedhuman transthyretin (TTR) amyloid-laden tissue, were subjected toantigen retrieval by incubation with CitraPlus (BioGenex, San Ramon,Calif.) for 30 min at 90° C. Peptide p5+14 was added to the tissue at ˜3μg/mL (30× molar excess over mAb) and incubated overnight at 4° C.Unbound reagent was removed by washing in PBST for 30 min. Tissues (withor without p5+14) were immunostained with a 3 μg/mL solution ofbiotinylated anti-peptide mAb (clones 4, 5, 12, or 13). Slides weredeveloped by addition of streptavidin-HRP (Vectastain Elite ABC kit,Vector Labs) followed by 3,3′-diaminobezidene (Vector Labs).

By way of positive control, a biotinylated-p5+14 peptide (without mAb)was used to directly stain TTR amyloid in this tissue (red arrows,below), as described above.

To confirm the presence and distribution of amyloid in the tissue aconsecutive slide was stained with Congo red. Briefly, tissues wereincubated in Congo red solution (0.8% w/v Congo red, 0.2% KOH w/v in 80%ethanol) for 1 h at RT. Sections were then washed in water andcounterstained by suspending in Mayer's hematoxylin for 2 min. Afterrinsing for 5 min in tap water the tissues were dehydrated in ethanol x2and Americlear before being coverslipped using a toluene-based mountingmedium. As shown in FIG. 6, the presence of amyloid was evidenced asgreen-red birefringent material in the Congo red-stained tissues whenviewed microscopically using cross-polarized illumination (whitearrows).

When directly biotinylated and added to tissue sections containing humanATTR amyloid, p5+14 peptide co-localizes with amyloid deposits which arealso observed in the Congo red-stained tissue section (FIG. 6). Whennon-biotinylated peptide p5+14 is added to the tissue sections and boundto the ATTR amyloid as a pre-targeting agent for the biotinylatedanti-peptide mAb, the amyloid was readily visualized in the tissuesection as brown deposits. Little or no “background” staining wasobserved. In contrast, when the biotinylated mAbs were added in theabsence of the pre-targeting p5+14 peptide there was no binding to theamyloid or healthy surrounding tissues (FIG. 7).

Discussion

These data indicate that the p5+14 peptide (or a similar variant) can beused to pre-target amyloid before addition of immunotherapeuticantibodies, such as the subcloned mAbs 4, 5, 12, or 13. The mAbs arecapable of binding directly to and targeting the amyloid-bound peptide,thereby triggering opsinization of the amyloid via a cellular immuneresponse (see below) that is capable of removing the tissue amyloiddeposits. In addition, the amyloid pre-targeting peptide can beradiolabeled and may be used as a molecular imaging agent, in additionto the first step in a pre-targeting anti-amyloid immunotherapyprotocol, as described in previous work (Wall et al. 2015, Molecules2015 Apr. 27; 20(5):7657-82. (PMID). Notably, because the pre-targetingpeptide p5+14, and similar reagents, have been shown to bind many typesof amyloid (regardless of the precursor protein from which the fibrilsare formed) pre-targeting immunotherapy using, e.g., peptide p5+14 witha suitable reactive mAb can be effective in many, if not all forms ofamyloidosis.

Example 2 Pre-targeting with Amyloid-Reactive Fusion Peptide

Visceral amyloidosis is characterized by the deposition of proteinfibrils in vital organs leading to dysfunction and death. At present,more than 27 different proteins have been identified as components ofamyloid fibrils in man, notably, immunoglobulin light chains (ALamyloid), transthyretin (ATTR), and serum amyloid protein A (AA).Immunotherapy, using amyloid fibril-reactive antibodies is beingdeveloped as a novel treatment. One antibody (mAb), designated 11-1F4has been shown to bind AL amyloid in patients with AL. Yet not allpatients were immunoreactive and this mAb does not bind ATTR or AAamyloid in vivo. Therefore, to enhance the utility of 11-1F4, we havedeveloped a synthetic bifunctional peptide (“peptope”—designated p66(SEQ ID NO:18) that combines a pan-amyloid-reactive peptide with a12-mer 11-1F4 epitope sequence. The p66 peptide was generated using thePeptide Synthesis and Purification described above in Example 1. UsingiTASSER (Iterative Threading ASSEmbly Refinement), we predicted twoprinciple structures based on the amino acid sequence of p66 (FIG. 8).

Interaction of murine 11-1F4 with peptide p66 and “natural” epitope

To show that the epitope part of p66 is not compromised by the presenceof the parent p5+14 parent sequence, we evaluated the interaction ofmurine 11-1F4 with peptide p66 and the “natural” epitope isolated from aκ4 immunoglobulin light chain, designated Len(1-22). More particularly,costar high-binding, 96-well, microplates were coated with 50 μl perwell of 0.83 mM p66 (peptope) peptide or V_(κ)4Len(1-22) peptide (the“natural epitope” of 11-1F4 present at the N-terminal of denatured kappa4 light chain proteins) overnight at 37° C. The plates were washed with1X solution of phosphate buffered saline with 0.05% (v/v) tween 20(PBST)—similar wash steps were performed between each step. As a“blocking step” the plates were incubated for 1 h at 37° C. with 200 μlof 1% (w/v) BSA in PBS per well. The murine 11-1F4 mAb binding wasassayed by titration from 100 nM (in BSAT-PBS, 0.05% (v/v) tween 20, 1%(w/v) BSA) as a starting concentration and diluted 1:2 across themicroplate and incubation for 1 h at 37° C. Biotinylated goat anti-mousesecondary antibodies (Sigma) were used at a 1:3000 dilution in BSAT.Europium-conjugated streptavidin (Perkin Elmer) was added (1:1000dilution of stock) as a detection medium and the plate incubated for 1 h37° C. Bound 11-1G4 was quantified following addition of enhancementsolution (Perkin Elmer) and the time-resolved fluorescence measuredusing a Victor 3 Wallac plate reader (Perkin Elmer).

We found that murine 11-1F4 bound both p66 and Len(1-22) when dried ontothe surface of a microplate (FIG. 9), thus showing that the epitope partof p66 is not compromised by the presence of the parent p5+14 parentsequence. The estimated affinity (EC50—concentration of mAb at 50%maximal binding) was estimated to be ˜0.5 nM for each peptide (FIG. 9).

Interaction of murine 11-1F4 with synthetic amyloid fibrils

To assess the interaction of 11-1F4 with synthetic amyloid fibrils, weevaluated the interaction of murine 11-1F4 with synthetic amyloidfibrils composed of the λ6 light chain Wil—associated with light chain(AL) amyloidosis, or Aβ(1-40), associated with Alzheimer's disease andcerebral amyloid angiopathy. More particularly, Costar high bindingplates were coated with 50 μl per well of 0.83 mM of synthetic amyloidfibrils composed of rVλ6Wil (AL fibrils) or Aβ(1-40) overnight at 37° C.The plates were washed with 1X solution of phosphate buffered salinewith 0.05% (v/v) tween 20 (PBST)—similar wash steps were performedbetween each step. As a “blocking step” the plates were incubated for 1h at 37° C. with 200 p.1 of 1% (w/v) BSA in PBS per well. The murine11-1F4 mAb was added from 100 nM (in BSAT), as a starting concentration,and diluted 1:2 across the microplate and incubation for 1 h at 37° C.Biotinylated goat anti-mouse secondary antibodies (Sigma) were used at a1:3000 dilution in BSAT. Europium-conjugated streptavidin (Perkin Elmer)was added (1:1000 dilution of stock) as a detection medium and the plateincubated for 1 h 37° C. Bound 11-1G4 was quantified following additionof enhancement solution (Perkin Elmer) and the time-resolvedfluorescence measured using a Victor 3 Wallac plate reader (PerkinElmer). We found that murine 11-1F4 bound both Wil and Aβ(1-40) fibrils,but did not saturate even at 0.1 μM 11-1F4 mAb (FIG. 10).

Effect of p66 on the interaction of murine 11-1F4 with synthetic amyloidfibrils

To asses the interaction of 11-1F4 antibodies with synthetic amyloidfibrils, we evaluated the interaction of murine 11-1F4 with syntheticamyloid fibrils composed of the λ6 light chain Wil—associated with lightchain (AL) amyloidosis, or Aβ(1-40), associated with Alzheimer's diseaseand cerebral amyloid angiopathy. More particularly, we assed theinteractions in the presence of p66. Costar high binding plates werecoated with 50 μl per well of 0.83 mM of synthetic amyloid fibrilscomposed of rVλ6Wil (AL fibrils) or Aβ(1-40) overnight at 37° C. Theplates were washed with 1X solution of phosphate buffered saline with0.05% (v/v) tween 20 (PBST)—similar wash steps were performed betweeneach step. As a “blocking step” the plates were incubated for 1 h at 37°C. with 200 μl of 1% (w/v) BSA in PBS per well. Peptide p66 (peptope)was added to the fibril-containing wells (100 p.1 of a 0.83 mM stocksolution) and the plate incubated for 1 h at 37° C. After a wash step toremove unbound peptope, the murine 11-1F4 mAb was added from 100 nM (inBSAT), as a starting concentration, and diluted 1:2 across themicroplate and incubation for 1 h at 37° C. Biotinylated goat anti-mousesecondary antibodies (Sigma) were used at a 1:3000 dilution in BSAT.Europium-conjugated streptavidin (Perkin Elmer) was added (1:1000dilution of stock) as a detection medium and the plate incubated for 1 h37° C. Bound 11-1G4 was quantified following addition of enhancementsolution (Perkin Elmer) and the time-resolved fluorescence measuredusing a Victor 3 Wallac plate reader (Perkin Elmer). We found that whenp66 was added to fibrils coated to the microplate well the reactivity ofthe 11-1F4 mAb was enhanced, particularly to the WI1 fibrils, but alsoto the Wil fibrils (FIG. 11).

Effect of BSA-blocking on p66 binding to synthetic fibrils

To asses the interaction of 11-1F4 antibodies with synthetic amyloidfibrils, we evaluated the interaction of murine 11-1F4 with syntheticamyloid fibrils composed of the λ6 light chain Wil—associated with lightchain (AL) amyloidosis, or Aβ(1-40), associated with Alzheimer's diseaseand cerebral amyloid angiopathy. More particularly, we assed theblocking ability of BSA on fibril binding. Costar high binding plateswere “blocked” by addition of 200 μl of 1% (w/v) BSA in PBS per well andincubation for 1 h at 37° C. Peptide p66 (peptope) was added to theblocked wells (100 μl of a 0.83 mM stock solution) and the plateincubated for 1 h at 37° C. After a wash step to remove unbound peptope,the murine 11-1F4 mAb was added from 100 nM (in BSAT), as a startingconcentration, and diluted 1:2 across the microplate and incubation for1 h at 37° C. Biotinylated goat anti-mouse secondary antibodies (Sigma)were used at a 1:3000 dilution in BSAT. Europium-conjugated streptavidin(Perkin Elmer) was added (1:1000 dilution of stock) as a detectionmedium and the plate incubated for 1 h 37° C. Bound 11-1G4 wasquantified following addition of enhancement solution (Perkin Elmer) andthe time-resolved fluorescence measured using a Victor 3 Wallac platereader (Perkin Elmer). We found that when p66 was added to BSA-blockedwells in the absence of fibrils, no peptide bound and no mAb reactivitywas observed (FIG. 12).

Binding of p66 and p5+14 to synthetic and naturally-occurring amyloid

To evaluate the binding of p66 and p5+14 to synthetic andnaturally-occurring amyloid, peptides p66 or p5+14 were radiolabeledwith iodine-125 (I-125, ¹²⁵1) using oxidation with chloramine T (1 mg/mlin water freshly made. Free 1-125 was removed from the reaction mixtureby size exclusion chromatography using a Sephadex G-25 solid phase and a0.1% (w/v) gelatin in PBS mobile phase. Fractions of ˜250 μl werecollected and the radioactivity in each measured using a gamma counter(Packard Cobra II auto-gamma counter). Peptide fractions with peakradioactivity were pooled and used for the “pull-down” assays.

For the pull down assay, ¹²⁵I-labeled peptide binding to murine: (m) AAand wild type (WT) liver homogenates (25 μl); rVλ6Wil (AL), Aβ(1-40) andislet amyloid polypeptide (IAPP) synthetic fibrils (25 μg), and;transthyretin-associated (ATTR), AL₇₈ 4-Cab and ALλ1-Ship human amyloidextracts (50 μg). The ¹²⁵I-peptides were prepared in PBST (0.15 M NaCl)or PBST with 1 M NaCl . Ten μl (˜5 ng, ˜100,000 counts per min [cpm]) ofthe ¹²⁵I-peptide solution was added to each test sample in a 200 μlvolume. The reaction mixtures were rotated for 1 h at RT, thencentrifuged twice at 16,000× g for 10 min. After each step thesupernatants were removed and collected in test tubes. The pellets,obtained following the second spin were resuspended in PBST. Theradioactivity in both the supernatant and pellet samples were measuredusing a Packard Cobra II auto-gamma counter. The bound peptide,expressed as % total was calculated according to:

Bound peptide (% total)=(Pellet cpm)/(Pellet cpm+Supernatant cpm)

We found that both peptide p66 (the 11-1F4 peptope) and p5+14 boundequally well to synthetic and naturally-occurring amyloid samples in0.15 M NaCl (FIG. 13A) and 1.0 M NaCl (FIG. 13B), indicating that thepresence of the 11-1F4 epitope sequence did not alter the binding to, oraffinity for, the amyloid samples.

Example 3 In vivo binding of radiolabeled ¹²⁵I-p66 peptope to AA amyloidin mice

Since there are no good mouse models of AL amyloidosis, we chose toinvestigate the reactivity of peptide p66 with systemic AA amyloidosisin a mouse model. Notably, the murine 11-1F4 mAb does not bind to AAamyloid in this mouse. Thus, this system will serve as an excellent toolto demonstrate induction of 11-1F4 reactivity by using the p66 peptide.Micro autoradiography was used to demonstrate uptake of the p66 peptide(labeled with iodine-125) in AA amyloid deposits in the mouse. Peptidep66 was produced and purified as described in Example 1. The p66 peptidewas radiolabeled with I-¹²⁵ as described above in Example 2. Otherdetailed methods are provided below.

Murine model of AA amyloidosis

Systemic visceral AA amyloidosis was induced in H2-L^(d)-huIL-6 TgBalb/c transgenic mice that constitutively express the humaninterleukin-6 transgene, by iv injection of 10 μg of purified, splenicAA amyloid (amyloid enhancing factor; AEF) in 100 μL of sterilephosphate-buffered saline (PBS). Peptide p66 was evaluated in mice at4-6 wk post AEF injection when amyloid load was significant.

SPECT/CT imaging of ¹²⁵I-p5+14 in AA and WT mice

Imaging was performed using WT or AA amyloid mice (n=3) that wereinjected with ˜5 μg of ¹²⁵I-p66, 125 μCi in the lateral tail vein. Afterthe appropriate uptake time (data for 4 and 72 h pi shown), mice wereeuthanized by isoflurane inhalation overdose. SPECT images were acquiredusing an Inveon trimodality imaging platform (Siemens PreclinicalSolution, Knoxville, Tenn.) running Inveon Acquisition Workplacesoftware (ver. 2.0). Low energy (^(125 I;) 25-45 keV) gamma photons wereacquired at each of 60, 16-sec projections with 90 mm of bed travel. A 1mm-diameter, 5-pinhole (Mouse Whole Body) collimator was used at 30 mmfrom the center of the field of view. Data were reconstructed post hoconto an 88×88×312 matrix with isotropic 0.50 mm voxels using a 3Dordered subset expectation maximization (OSEM) algorithm (8 iterations;6 subsets).

CT data were acquired using an x-ray voltage biased to 80 kVp with a 500mA anode current, with 4×4 binning. A 225 msec exposure was used, and360, 1-degree projections were collected. The data were reconstructedusing an implementation of the Feldkamp filtered back-projectionalgorithm onto a 512×512×1296 matrix with isotropic 0.106 mm voxels.SPECT and CT datasets were automatically co-registered and visualized byusing the Inveon Research Workplace visualization software package(Siemens Preclinical Solution, Knoxville, Tenn.). Mice wereadministered, IP, ˜300 μL of Iohexol CT contrast agent diluted 1:1 insterile PBS, 5 min before the imaging data were acquired.

Biodistribution measurements

Samples of liver, spleen, pancreas, kidneys, small and large intestines,stomach and heart were harvested post mortem from every mouse undergoingimaging with ¹²⁵I-p66. A sample of each was placed into a tared, plasticvial, weighed and the ¹²⁵I radioactivity measured using an automatedWizard 3 gamma counter (1480 Wallac Gamma Counter, Perkin Elmer). Thebiodistribution data were expressed as % injected dose/g tissue (%ID/g). In addition, samples of each tissue were fixed in 10%buffered-formalin for 24 h and embedded in paraffin for autoradiography.

Micro-autoradiography and CR staining

For autoradiography, 6-μm-thick sections were cut from formalin-fixed,paraffin-embedded blocks, containing tissues from mice that had received¹²⁵I-p66. The sections were placed on Plus microscope slides (FisherScientific), dipped in NTB-2 emulsion (Eastman Kodak), stored in thedark, and developed after a 4 day exposure. Each section wascounterstained with hematoxylin and eosin. Detection of amyloid wasachieved in consecutive tissue sections by staining with an alkalineCongo red solution (0.8% w/v Congo red, 0.2% w/v KOH, 80% ethanol) for 1h at room temperature followed by conunterstain with Mayer's hematoxylinfor 2 min.

All tissue sections were examined using a Leica DM500 light microscopefitted with cross-polarizing filters (for Congo red). Digitalmicroscopic images were acquired using a cooled CCD camera (SPOT;Diagnostic Instruments).

Results and Discussion

Radiolabeled (¹²⁵1) p66 injected into mice with systemic AA amyloidosisspecifically bound the amyloid deposits as evidenced by the depositionof black silver grains in the autoradiographs (indicative of ¹²⁵I-p66)at the sites of amyloid deposition, seen as green-gold birefringence inthe Congo red-stained tissues (FIG. 14). Further, micro autoradiographydemonstrated that ¹²⁵I-p66 peptide does not bind healthy tissues (FIG.15). More particularly, radiolabeled (¹²⁵I) p66 injected into healthy WTmice did not bind to any tissue that was studied, as evidenced by theLACK of black silver grains in the autoradiographs (indicative of¹²⁵I-p66) (FIG. 15). Lastly, as shown in FIG. 16, reactivity of ¹²⁵I-p66with amyloid in vivo, notably the liver, spleen, pancreas, andintestines was confirmed by SPECT/CT imaging and tissue biodistributionmeasurements. The reactivity with amyloid in vivo was sufficientlystable that the amyloid in the liver could be readily visualized, bySPECT imaging, at least 72 h post injection of the peptide (FIG. 16A andFIG. 16B).

Example 4 Pre-targeting of ¹²⁵I-11-1F4 to Systemic AA Amyloid in vivo

The murine mAb 11-1F4 mAb does not efficiently bind to AA amyloid in themurine model of AA amyloidosis. Therefore, we sought to demonstratespecific AA amyloid binding in mice by ¹²⁵I-11-1F4 by using peptope p66as a pre-targeting agent. Micro autoradiography, combined withimmunohistochemical detection of amyloid-bound p66 in the mice, was usedto demonstrate ¹²⁵I-11-1F4 and p66 peptope binding to AA amyloiddeposits in the mouse. As a control, the mice received an IV injectionof peptide p5+14, instead of p66. Peptide p66 was produced and purifiedas described in Example 1. The p66 peptide was radiolabeled with I¹²⁵ asdescribed above in Example 2. Other detailed methods are provided below.

Murine model of AA amyloidosis

Systemic visceral AA amyloidosis was induced in H2-L^(d)-huIL-6 TgBalb/c transgenic mice that constitutively express the humaninterleukin-6 transgene, by iv injection of 10 μg of purified, splenicAA amyloid (amyloid enhancing factor; AEF) in 100 μL of sterilephosphate-buffered saline (PBS). Peptide p66 pre-targeting of¹²⁵I-11-1F4 was evaluated in mice at 3-4 wk post AEF injection whenamyloid load was modest.

In vivo Pre-targeting

Three cohorts of 3 mice, each received ˜400 μg of unlabeled peptide p66,and a second group of 3 cohorts were given peptide p5+14 as a control.Twenty-four hours after the peptide injection all mice were administered˜150 μCi (˜20 μg) of ¹²⁵I-11-1F4 IV in the lateral tail vein. One groupof p66 mice (n=3) and one group of p5+14 control mice (n=3) wereeuthanized at 24, 48 and 72 h post injection of 11-1F4 mAb and theorgans harvested at necropsy for fixation, followed bymicroautoradiographic and immunohistochemical analyses.

Micro-autoradiography and Congo Red staining

For autoradiography, 6-μm-thick sections were cut from formalin-fixed,paraffin-embedded blocks, containing tissues from mice that had received¹²⁵I-p66. The sections were placed on Plus microscope slides (FisherScientific), dipped in NTB-2 emulsion (Eastman Kodak), stored in thedark, and developed after a 4 day exposure. Each section wascounterstained with hematoxylin and eosin. Detection of amyloid wasachieved in consecutive tissue sections by staining with an alkalineCongo red solution (0.8% w/v Congo red, 0.2% w/v KOH, 80% ethanol) for 1h at room temperature followed by counterstain with Mayer's hematoxylinfor 2 min.

Immunohistochemistry

Formalin-fixed paraffin embedded tissue sections, from mice treated withp66 peptope or peptide p5+14 were subjected to antigen retrieval usingcitrate buffer (pH 6; Dako) 30 min at 90° C. The tissue was then blockedwith hydrogen peroxide, casein, and avidin and biotin, per manufacturersinstructions. The biotinylated peptide-reactive mAb (clone 12-3[described above]) was then added (1.6 μg/mL in PBS) an the samplesincubated for 2 h at RT, 0/N at 4° C., followed by another 2 h period atRT. After washing the tissue, slides were developed by adding Vector ABCElite, for 40 min at RT, followed by Vector DAB.

The presence of macrophages was detected by staining with mAb Iba-1(1:8000 diln) followed by addition of biotinylated rabbit anti-mousesecondary reagent (Vector Rabbit Elite kit). The slides were developed,as described above. All tissue sections were examined using a LeicaDM500 light microscope fitted with cross-polarizing filters (for Congored). Digital microscopic images were acquired using a cooled CCD camera(SPOT; Diagnostic Instruments).

Results and Discussion

At 24 h post injection of 11-1F4 mAb into AA mice pre-targeted withpeptope p66, ¹²⁵-11-1F4 localizes with p66 (FIG. 17). More particularly,brown coloration in the immunohistochemical stain is indicative of thepresence of peptide p66 associated specifically with AA amyloid in thetissues (FIG. 17). Black punctate coloration in the autoradiographs isindicative of the presence of ¹²⁵-11-1F4, which is seen exclusivelyco-localized with the p66-coated AA amyloid (FIG. 17).

In contrast, evaluation of mice at 24 h post injection of 11-1F4 mAbinto AA mice pre-targeted with p5+14 control peptide did not showco-localization (FIG. 18). That is, despite the presence of brownp5+14-coated AA amyloid in all tissues evaluated, there was littleevidence of ¹²⁵1-11-1F4 co-localized with the amyloid, as evidenced bythe absence of black silver grains in the microautoradiographs (FIG.18).

Lastly, liver macrophages in AA mice at 72 h post injection of 11-1F4mAb pre-injected with p66 or p5+14 showed induced macrophageinfiltration (FIG. 19). More particularly, brown coloration wasassociated with the Iba-1 positive macrophages in the mouse liver. Thesepreliminary data suggest that the combination of p66 with 11-1F4 (FIG.19 (upper)) in AA mice induced macrophage infiltration into the liverand clustering of macrophages around amyloid deposits to a greaterdegree that p5+14 in conjunction with the 11-1F4 mAb (FIG. 19 (lower)).

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1-46. (canceled)
 47. A method of targeting an amyloid deposit forclearance, comprising: contacting an amyloid deposit with anamyloid-reactive peptide that binds an amyloid deposit; and, contactingthe amyloid-reactive peptide with an antibody or functional fragmentthereof that binds the amyloid-reactive peptide, wherein contacting theamyloid-reactive peptide with the antibody that binds theamyloid-reactive peptide targets the amyloid deposit for clearance. 48.The method of claim 47, wherein targeting the amyloid deposit forclearance results in clearance of the amyloid deposit.
 49. The method ofclaim 47, wherein an epitope is fused to the amyloid-reactive peptideand wherein the antibody or functional fragment thereof binds theepitope.
 50. The method of claim 49, wherein binding of the antibody orfunctional fragment thereof to the fused epitope results in clearance ofthe amyloid deposit from the subject.
 51. The method of claim 49,wherein the epitope comprises an 11-1F4 antibody-binding motif.
 52. Themethod of claim 51, wherein the epitope is at least 95% identical to theamino acid sequence set forth as SEQ ID NO:22 or SEQ ID NO:23.
 53. Themethod of claim 47, wherein the antibody or functional fragment thereofis an amyloid-reactive antibody or functional fragment thereof.
 54. Themethod of claim 47, wherein the amyloid-reactive peptide binds to one ormore amyloids comprising AA, AL, AH, ATTR, Aβ2M, ALect2, Wild type, TTR,AApoAI, AApoAII, AGel, ALys, ALect2, Afib, ACys, ACal, AMedin, AIAPP,APro, Ahs, APrP, or Afβ amyloid.
 55. The method of claim 47, wherein theamyloid-reactive peptide is at least 95% identical to the amino acidsequence set forth as any one of SEQ ID NO:1 to SEQ ID NO:17.
 56. Themethod of claim 49, wherein the fused amyloid-reactive peptide andepitope comprise an amino acid sequence that is at least 95% identicalto the amino acid sequence set forth as SEQ ID NO:18, SEQ ID NO:19, orSEQ ID NO:20.
 57. The method of claim 47, wherein the antibody is amonoclonal antibody that binds one or more of the amyloid-reactivepeptides having an amino acid sequence at least 95% identical to one ormore of the amino acid sequences set forth as SEQ ID NOS:1, 5, 8, 12,13, 15, 16, or
 17. 58. A method for clearing an amyloid deposit in asubject, comprising: administering to the subject an amyloid-reactivepeptide and an antibody or functional fragment thereof that binds theamyloid-reactive peptide, wherein the amyloid-reactive peptide binds anamyloid deposit and wherein the administration of the amyloid-reactivepeptide and the antibody or functional fragment thereof to the subjectresults in clearance of the amyloid deposit in the subject.
 59. Themethod of claim 58, wherein an epitope is fused to the amyloid-reactivepeptide and wherein the antibody or functional fragment thereof bindsthe epitope.
 60. The method of claim 59, wherein the epitope comprisesan antibody binding motif and wherein the antibody binding motif bindsto an amyloid deposit.
 61. The method of claim 59, wherein the epitopecomprises an 11-1F4 antibody-binding motif.
 62. The method of claim 61,wherein the epitope is at least 95% identical to the amino acid sequenceset forth as SEQ ID NO:22 or SEQ ID NO:23.
 63. The method of claim 59,wherein the fused amyloid-reactive peptide and epitope comprise an aminoacid sequence at least 95% identical to the amino acid sequence setforth as SEQ ID NOS:18, SEQ ID NO:19, or SEQ ID NO:20.
 64. The method ofclaim 58, wherein the amyloid-reactive peptide is at least 95% identicalto the amino acid sequence set forth as any one of SEQ ID NO:1 to SEQ IDNO:17.
 65. A method for treating amyloidosis in a subject, comprising:administering to a subject an effective amount of an amyloid-reactivefusion peptide, wherein the amyloid-reactive fusion peptide comprises anamyloid-reactive peptide that binds to an amyloid deposit and an epitopefused to the amyloid-reactive peptide that binds an antibody; andadministering to the subject an effective amount the antibody orfunctional fragment thereof.
 66. The method of claim 65, wherein bindingof the antibody or fragment thereof to the amyloid-reactive fusionpeptide results in clearance of the amyloid deposit.
 67. The method ofclaim 65, wherein the amyloid reactive-peptide of the amyloid-reactivefusion peptide is at least 95% identical to the amino acid sequence setforth as any one of SEQ ID NO:1 to SEQ ID NO:17.
 68. The method of claim65, wherein the amyloid-reactive peptide of the amyloid-reactive fusionpeptide binds to one or more amyloid deposit types comprising AA, AL,AH, ATTR, Aβ2M, ALect2, Wild type, TTR, AApoAI, AApoAII, AGel, ALys,ALect2, Afib, ACys, ACal, AMedin, AIAPP, APro, Alns, APrP, or Aβ. 69.The method of claim 65, wherein the antibody or functional fragmentthereof is reactive to one or more of the peptides having an amino acidsequence set forth as SEQ ID NO:18 or SEQ ID NO:19 or SEQ ID NO:20. 70.The method of claim 65, wherein the epitope is an epitope of an 11-1F4antibody.
 71. The method of claim 70, wherein the epitope has an aminoacid sequence set forth as one or more of SEQ ID NO:22 or SEQ ID NO:23.